JP2010263904A - Viral vector - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a useful and safe viral vector. <P>SOLUTION: There is provided a multicistronic retroviral vector genome comprising a first nucleotide sequence upstream of an internal regulatory element, such that the level of genomic RNA available for packaging in the absence of rev, or a functional equivalent thereof, is increased. There is also provided a vector genome comprising a nucleotide sequence which is operably linked to a viral LTR and is upstream of an internal regulatory element. The nucleotide sequence is upstream of an internal promoter or IRES, and enhances the level of genomic RNA available for packaging in the absence of rev. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

The present invention relates to viral vector genomes, production systems, and viral particles comprising them, and in particular, but not exclusively, their use in therapy. The invention further relates to a method for identifying a targeting moiety and / or a binding modulating moiety. The present invention further relates to a vector that can be used in the identification method of the present invention.

Retroviral vector systems, such as, for example, lentiviral vector systems, have been proposed as delivery systems for transporting nucleotides of interest to one or more sites of interest. In fact, the concept of using viral vectors for gene therapy is well known (Verma and Somia (1997) Nature 389: 239-242). The retroviral genome includes accessory genes such as the rev gene, tat gene, vif gene, nef gene, vpr gene, or S2 gene. Such deletion of auxiliary genes is very advantageous, especially when retroviral vector systems are used for gene therapy. First, it allows the production of vectors that do not contain genes normally associated with diseases (eg, HIV) in retroviral infection. Secondly, deletion of the auxiliary gene allows the packaging of additional heterologous DNA in the vector. Third, genes with unknown functions such as dUTPase and S2 can be removed, thereby reducing the risk of causing undesirable effects. Applicants have previously taught a method for removing many auxiliary genes, for example in Applicants' WO 98/17815. Further, in Applicants' WO 99/45126, Applicants describe codon optimization of gag-pol sequences as a means to overcome Rev / RRE requirements for transport and enhance RNA stability. is doing.
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However, there remains a need to provide strategies for providing useful and safe viral vectors and efficient means for producing them. The present invention addresses these problems and particularly advantageously aims to provide a safer system that does not require viral accessory genes such as rev in the production of viral vector particles used in therapy, or their production.

Thus, in one aspect of the invention, an internal regulatory element such as a promoter or IRES (Internal Ribosome Entry Site) so that the level of genomic RNA available for packaging is increased in the absence of rev or its functional equivalent. A multicistronic retroviral vector genome comprising a first nucleic acid sequence upstream is provided.

Accordingly, Applicants have further found here that viral vectors that are independent of rev can be provided without adversely affecting the viral titer.

Further according to the invention, the lentivirus is a multicistronic vector, the vector genome contains a nucleotide sequence operably linked to the viral LTR, said nucleotide sequence upstream of the internal regulatory element Retroviral vectors such as are provided.

Further according to the invention, the vector is multicistronic and the vector genome contains a nucleotide sequence operably linked to the viral LTR, said nucleotide sequence being upstream of an internal promoter or IRES, rev In the absence, a retroviral vector such as a lentivirus is provided that can increase the level of genomic RNA available for packaging. This nucleotide sequence preferably encodes a polypeptide or fragment thereof.

Preferably, the vector genome is 2, 3, or 4 cistronic.

Preferably, the vector is in the form of a lentiviral vector genome.

Preferably, the first nucleic acid sequence is an open reading frame (ORF), or part thereof.

In one embodiment, the first nucleic acid sequence is a reporter moiety, or a selectable marker.

Preferably, the ORF is downstream of the viral LTR.

Preferably, the ORF is operably linked to the LTR.

Preferably, the nucleic acid sequence encoding the auxiliary gene rev or a functional equivalent thereof has been disrupted or removed from the vector genome such that the auxiliary gene cannot encode a functional auxiliary protein, or It is not supplied by a transformer from another source.

In other embodiments, the first nucleic acid sequence encodes a modulator gene.

Preferably, the modulator gene is selected from tetracycline repressors, such as TetR, and tetracycline-regulated transactivators, such as tTA and rtTA.

Preferably, the tetracycline inhibitor is a codon that is optimized for expression in mammalian cells.

Preferably, the tetracycline inhibitor is bound to a nuclear localization signal.

In a preferred embodiment, the vector further comprises one or more NOIs downstream of the internal promoter or IRES and optionally operably linked thereto.

Preferably, the NOI produces a therapeutic effect.

In one embodiment, the one or more NOIs are operably linked to a tetracycline operator.

In other embodiments, the vector further comprises a tetracycline repressor downstream of the internal promoter or IRES.

Preferably, the vector is derived from HIV-1, HIV-2, SIV, FIV, BLV, EIAV, CEV, or Bisnarlent virus.

Preferably, the vector is derived from a non-primate lentivirus.

Preferably the vector genome comprises a cPPT sequence.

Preferably, the vector genome includes post-transcriptional regulatory elements or translation elements.

Preferably, the ATG motif of the gag packaging signal of the wild type lentiviral vector genome is an ATTG motif.

Preferably, the distance between the R regions of the vector genome is substantially the same as the distance in the wild type lentiviral vector.

Preferably, the 3'U3 region of the vector genome comprises viral and / or eukaryotic promoter sequences.

Preferably, the 3'U3 region of the vector genome comprises a wild type virus and / or a eukaryotic promoter.

Preferably, the viral promoter is an EIAV or MLV U3 region.

Preferably, the promoter is a self-inactivating LTR.

Thus, according to the present invention, a lentiviral vector is provided wherein the vector has the following: The ATG motif of the gag packaging signal of the wild type lentiviral vector is an ATTG motif; the distance between the R regions of the lentiviral vector Is substantially the same as the distance in the wild-type lentiviral vector; the 3′U3 region of the lentiviral vector contains viral and / or eukaryotic promoter sequences. Preferably, the 3'U3 region may comprise an EIAV or MLV U3 region, or other viral or eukaryotic promoter sequences. In one embodiment, the 3'U3 region can comprise a wild type virus or a eukaryotic promoter.

According to another aspect of the present invention, there is provided a retroviral vector production system for producing retroviral-derived vector particles, the system comprising the vector genome, gag and pol protein, env protein of any one of the claims Or a set of nucleic acid sequences encoding a component of the vector containing functional alternatives thereof, wherein the vector genome is multicistronic and is available for packaging in the absence of rev, Alternatively, a first nucleic acid sequence is included upstream of an internal regulatory element, such as a promoter or IRES, so as to increase the level of retroviral analog auxiliary genes from which the particles are derived.

Preferably, in this system, the nucleic acid sequence encoding the auxiliary gene rev, or a similar auxiliary gene of the retrovirus from which the particle is derived, is disrupted such that the auxiliary gene cannot encode a functional auxiliary protein. Or has not been supplied in transformers from other sources.

Preferably, in this system, the nucleic acid sequence encoding at least one auxiliary gene vpr, vif, tat and nef of the retrovirus from which the particle is derived or a similar auxiliary gene is also encoded by the auxiliary gene. It has been destroyed so that it cannot be removed or removed from the system.

Preferably, the vector is derived from a lentivirus.

Preferably, the vector is derived from HIV-1, HIV-2, SIV, FIV, BLV, EIAV, CEV, or Bisnarlent virus.

Preferably, the vector is derived from a non-primate lentivirus.

Preferably, the set of nucleic acid sequences encoding the components of the vector comprises three DNA constructs encoding the RNA genome of the vector, the Gag and Pol proteins, and the Env protein, or a functional alternative thereof.

According to another aspect of the present invention, there is provided a DNA construct for use in the system of the present invention, said DNA construct encoding a packageable RNA vector genome according to the present invention.

According to another aspect of the present invention, there is provided a set of DNA constructs for use in the system of the present invention, comprising a DNA construct according to the present invention and a DNA construct encoding a Gag and Pol protein, or a functional alternative thereof. .

Preferably, the set further comprises a DNA construct encoding the Env protein, or a functional alternative thereof.

According to another aspect of the present invention, a method for preparing retroviral vector particles comprising introducing a set of nucleic acid sequences or DNA constructs of the present invention into a host cell, obtaining a retroviral vector particle, and the present invention. Retroviral vector particles produced by the system or method according to are provided.

According to another aspect of the present invention, there is provided a retrovirus-derived vector particle comprising an RNA genome of a vector, a Gag and Pol protein, and an Env protein, or a functional alternative thereof, wherein the vector genome is according to the present invention. The

In other embodiments, cells transduced with the retroviral vector particles or DNA constructs of the invention are provided.

In another aspect of the invention there is provided a pharmaceutical composition comprising a vector, system, particle or cell according to the invention together with a pharmaceutically acceptable carrier or diluent.

In another aspect of the invention, there is provided the use of a retroviral vector particle or DNA construct or cell of the invention for preparing an agent that delivers NOI to a target site in need thereof.

In another aspect of the present invention, a delivery system is provided for use in medicine that is in the form of a retroviral vector particle or DNA construct or cell of the present invention.

In complex organisms, each gene product is often part of a network or pathway that can represent only a single step in a series of complex intracellular or extracellular interactions, and the function of the gene product is It is thought that it depends on the cellular status. Thus, by understanding the function of a gene product in a cell and connecting it to a biological process, a candidate for a pharmacological manipulation, ie a therapeutic target, can be identified. Clearly, establishing the value of any potential gene target requires finding and confirming its role in the disease process. Ideally, the unique role of the target candidate gene is elucidated in disease-related regulatory pathways. However, the gene regulatory mechanism at the cellular level is very complex and reflects the complex nature of the signaling pathway. The elucidation of the role of potential target genes is further complicated by cell type specificity issues. That is, the function of the potential gene target of a cell type, or even one species, may be different from other cell types.

Another problem is the elucidation of the relationship between the target gene, its clinical applicability, and the resulting gene product. If there is a unique “causal” relationship in a biological regulatory system, the function of a potential target gene may be relatively easily elucidated. As an example, several highly successful biopharmaceutical products, including insulin, erythropoietin (EPO), and granulocyte colony stimulating factor (GCSF) act through the ability to modulate such relationships. However, the unique “causal” relationship is an exception and is not common. Furthermore, the development of receptor ligands as therapeutic agents is complex due to ligand duplication and cell type specificity. Examples include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), interleukins (IL-10, IL-12, IL-18, etc.), transforming growth factor beta (TGF-b), and Ligands such as tumor necrosis factor (TNF) can exhibit duplication, cell type specificity, and non-linear dose dependence, which can lead to failure of clinical trials of these potential therapeutic agents.

Previous attempts to address the challenges posed by the complexity of cell regulation have been directed to the development of techniques that help elucidate and understand complex biological networks. These techniques include, but are not limited to, methods for identifying individual pathway components, including proteomics, multi-hybrid systems, expression cloning, and DNA chips. These methods are applied by inferring function from direct and indirect associations between individual signaling components or by assuming function from event correlation. However, such methods are cumbersome, time consuming and often result in ambiguous results.

As an example, a novel measuring the interaction of G protein with a G protein coupled receptor (GPCR) using the concept of a “chip” that immobilizes the receptor on a solid surface to allow easy detection of activity A new assay has been developed (see Bieri et al., Nat Biotechnol (1999) 17: 1105-1108). An important feature of this system is that the receptor-G protein complex is immobilized on the sensor chip in a functional form. That is, patterned immobilization of GPCRs on the chip surface and direct detection of functional responses. A possible application of this approach is the screening of small molecules or peptides that can activate known or orphan receptors. However, this screen has the disadvantage that the antagonist is not recognized because the receptor must be active and result in the release of G protein. In addition, technical problems associated with chip-based screening include determining whether a GPCR is still functional and accessible during high-throughput screening.

Other approaches to address the complexity of signal transduction pathways use high-throughput techniques such as high-throughput screening (HTS) to screen drug candidates for direct efficacy against specific disease-related gene sites. Engineered cell-based assays have been used. Such screening is based on the activity of reporter genes introduced into cells that directly indicate the activity of the gene at any regulatory site in the genome. As an example, cell-based assays must perform most aspects of function when the target GPCR is cell-based, assembly into the plasma membrane with the appropriate ligand-binding configuration, and ligand inside the cell. It is useful in the identification of target GPCRs since it takes place in a natural situation involving the transmission of binding signals.

Several cell-based screening methods using retroviral vectors have also been developed. As an example, Li et al., Nucleic Acids Research (2000) 28 (13): 2605-2612 discloses a functional genomic approach using a hairpin ribozyme gene library with randomized target recognition sequences constructed in retroviral vectors. is doing. As a further example, Beger et al., PNAS (2001) 98 (1) 130-135 teaches that retroviral vectors can be used to produce ribozyme library vectors to identify cellular genes that regulate BRCA1 expression. ing. However, retroviral vector screening has drawbacks because retroviral vectors cannot transduce non-dividing cells such as primary differentiated non-dividing cells.

As a result, several target moieties have been identified to date using standard screening techniques, but these techniques do not allow selection of candidate moieties specific to the target host cell. In particular, these screening techniques do not allow the development of screening techniques adapted to specific target cells. Furthermore, in particular, these screening techniques stop differentiation and do not allow selection of specific candidate moieties for non-dividing target host cells.

Accordingly, an object of this aspect of the invention is to provide a cell-based screen that identifies unknown components of cellular response pathways that may be useful as potential drug targets for therapeutic intervention.

This aspect of the invention is further directed to the identification of components that can interact with these targets that may be useful as potential therapeutic agents.

This screen further facilitates the detection of altered cellular responses to responses that normally occur in a given environmental or disease state.

This aspect of the invention
(I) adapt the screening to a particular cell or disease state;
(Ii) identifying new targeting moieties and / or binding modulating moieties by combination with screening that can be performed using slow dividing cell types such as non-dividing cell types or primary terminally differentiated non-dividing cells,
(Iii) identifying possible target and therapeutic sequences using dual purpose screening;
(Iv) It is advantageous since it provides a means for screening using a route that is particularly suitable for in vivo conditions.

Other advantages are set forth in and will be apparent from the following description.

This aspect of the invention allows for the selection of candidate moieties and provides an improved method for screening candidate target moieties and / or binding modulator moieties using screening systems that are suitable for non-dividing cells or slow dividing cells. The purpose is to provide.

Thus, the present invention uses an improved screening system that allows the selection of candidate target moieties and screens for binding target modulating moieties using vectors suitable for candidate target moieties and / or non-dividing or slow dividing cells. It aims to provide a method.

According to this aspect of the invention, there is provided a method for identifying a cell response moiety and / or a binding modulating moiety, the method comprising:
(I) providing a target non-dividing or slowly dividing cell comprising a group of NOIs encoding a plurality of modulating moieties;
(Ii) optionally exposing the cell to a biological response modifier;
(Iii) detecting a phenotypic difference between a target non-dividing or slow-dividing cell encoding or expressing at least one modulating moiety and a control cell in which no modulating moiety is present, The phenotypic difference includes the result of binding of the modulating moiety to the cell response moiety, and (iv) recovering the cell response moiety and / or the binding modulating moiety.

Examples of important non-dividing target cells include neurons, certain cells of the immune system, certain epithelial cells, hepatocytes, and islets of Langerhans. Furthermore, many cells that divide but divide very slowly exist, for example, in the human body. These slow dividing cells are also poor recipients of genes delivered by, for example, MLV-based vectors, which include cells in solid tumors and some non-neuronal cells in the brain . For example, most cells within a tumor are not dividing at any time or are dividing slowly.

In addition, cells that do not normally divide in the body can divide slowly during culture, such as primary and neuronal cells in culture. Therefore, the present invention can be applied not only to non-dividing cells but also to slowly dividing cells.

In a particularly preferred embodiment, a group of NOIs is constructed with a viral vector. In particular, the viral vector can be selected from the group consisting of a lentiviral vector, an adenovirus vector, an adeno-related vector, a herpes vector, a poxvirus vector, a parvovirus, and a baculovirus vector. The use of lentiviral vectors is particularly preferred.

Preferably, the viral vector of the present invention has a minimal viral genome.

As used herein, the term “minimal viral genome” refers to non-essential elements to provide the required functionality to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell. It means a viral vector that has been manipulated to remove and retain the essential elements.

Preferably, the viral vector having the minimal viral genome is a lentiviral vector.

In other embodiments of the invention, the library of candidate modulating moieties is introduced into an adeno-associated virus (AAV) vector.

In other embodiments of the invention, the library of candidate modulating moieties is introduced into a poxvirus vector.

In other embodiments of the invention, the library of candidate modulating moieties is introduced into a herpesvirus vector.

In other embodiments of the invention, the library of candidate modulating moieties is introduced into a baculovirus vector.

In other embodiments of the invention, a library of candidate modulating moieties is introduced into a parvovirus vector (see Kestler et al., 1999, Human Gene Ther 10 (10): 1619-32).

According to a second aspect of the invention, there is provided a method for identifying a targeting moiety and / or a binding modulating moiety, comprising:
Optionally exposing the cell to a biological response modifier, providing a target cell encoding a plurality of modulating moieties and comprising a group of NOIs constructed in a lentiviral vector;
Detecting a phenotypic difference between a target cell encoding or expressing at least one modulating moiety and a control cell in which no modulating moiety is present, the phenotypic difference being modulated The step resulting from the binding of the moiety to the target moiety, and the step of recovering the target moiety and / or the binding modulating moiety.

Lentiviral vectors of the present invention include HIV vectors (eg, HIV-1 and HIV-2 vectors), primate lentiviral vectors such as SIV vectors, and non-primate lentiviral vectors. Primate lentiviral vectors have several drawbacks that may limit their therapeutic application to certain diseases. For example, HIV-1 has the disadvantage that it is a human pathogen with potentially oncogenic proteins and sequences. The introduction of vector particles produced in packaging cells that express HIV gag-pol introduces these proteins into the individual and risks seroconversion. Thus, in a particularly preferred embodiment, the lentiviral vector is a non-primate lentiviral vector such as EIAV, FIV, BIV, CAEV, or MVV, with EIAV being particularly preferred. Non-primate lentivirus-based vectors do not introduce HIV protein into an individual.

In one embodiment, the method further comprises isolating and / or sequencing the cell response portion and / or the binding modulating portion.

In a preferred embodiment, the phenotypic difference is a reporter gene transcription or expression difference. Preferably, the reporter gene is operably linked to a regulatory moiety.

In other preferred embodiments, the phenotypic difference is modulation of the binding of at least one polypeptide to another polypeptide.

Preferably, the phenotypic difference is the appearance or absence of a detectable phenotype.

Preferably, the group of NOIs is selected from the group consisting of a ribozyme library, an antisense library, and a cDNA library. Alternatively, the NOI can be a polynucleotide sequence that encodes a polypeptide selected from or using an antibody library. The antibody library is preferably an intracellular antibody (or “intracellularly expressed antibody”) library.

In preferred embodiments, the NOI is constitutively expressed. In other embodiments, the expression of NOI is under the control of a regulatable promoter.

In preferred embodiments, any biological response modifier mimics a disease state.

Preferably, the lentiviral vector is an EIAV vector.

In preferred embodiments, the target cell comprises a reporter moiety operably linked to a regulatable moiety. In one embodiment, the adjustment moiety is an adjustment element. Regulatory elements include ischemia-like response elements (ILRE), such as hypoxia response elements (HRE). In other embodiments, the regulatory element is a promoter.

According to another aspect of the invention there is provided a targeting moiety and / or a binding modulating moiety identified by a method according to any one of the preceding claims.

According to a third aspect of the present invention there is provided a composition comprising an identified targeting moiety and / or binding modulating moiety according to the present invention for use in medicine.

According to a fourth aspect of the invention, there is provided a method of treating a disease in a subject, the method comprising administering to the subject an identified targeting moiety and / or binding modulating moiety according to the invention. .

According to a fifth aspect of the present invention, the use of an identified target moiety and / or binding modulator moiety according to the present invention in the preparation of a medicament for treating a disease associated with a cell response moiety and / or a binding modulating moiety. Is provided.

In another aspect of the invention,
(I) an optional first nucleotide sequence encoding a selectable marker;
(Ii) one or more optional second nucleotide sequences, each independently encoding a marker or modulator gene, and (iii) can bind to the nucleotide sequence and effect its cleavage directly or indirectly A library vector comprising a third nucleotide sequence operably linked to a promoter encoding a gene product can be used;
(I), (ii), and (iii) are operably linked to regulatory sequences capable of directing transcription of (i), (ii), and (iii) as continuous RNA molecules in the host.

In other embodiments,
(A) a first nucleotide sequence encoding a modulator gene;
(B) a second nucleotide sequence encoding a detectable marker gene and a promoter operably linked to the nucleotide sequence that the first nucleotide sequence modulates; and (c) a nucleotide sequence of interest. A targeting vector can be used,
(A), (b), and (c) are operably linked to regulatory sequences capable of directing transcription of (a), (b), and (c) as continuous RNA molecules in the host.

The screening method of the present invention can be used for various applications. For example, a cDNA library can be screened for proteins that interact with the protein of interest (functional genomics) to aid in the analysis of protein interaction pathways.

A library of randomized sequences, particularly partially randomized sequences, is directed evolution screening to “evolve” proteins or nucleotides with improved binding affinity or catalytic activity compared to wild type protein / nucleotide Can be used. For example, a randomized library can be used to select DNA binding proteins that have a specific specificity for a given sequence. Another application of this approach is affinity maturation of antibodies.

Other applications include screening libraries encoding peptides that identify peptide inhibitors or activators of proteins, such as enzymes.

Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples. In general, the techniques described herein are well known in the art, but in particular, Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Short Protocols in Molecular Biology (1999), 4th edition, Reference can be made to John Wiley & Sons, Inc. (and the full version of Current Protocols in Molecular Biology).

Retroviruses and lentiviruses As noted above, the concept of using viral vectors for gene therapy is well known (Verma and Somia (1997) Nature 389: 239-242).

There are many retroviruses. In this application, the term “retrovirus” includes mouse leukocyte virus (MLV), human immunodeficiency virus (HIV), equine infectious anemia virus (EIAV), mouse mammary adenocarcinoma virus (MMTV), rous sarcoma virus (RSV). ), Fujinami sarcoma virus (FuSV), Moloney mouse leukocyte virus (Mo-MLV), FBR mouse osteosarcoma virus (FBR MSV), Moloney mouse sarcoma virus (Mo-MSV), Ebelson mouse leukocyte virus (A-MLV), Avian myelocytoma virus-29 (MC29), and avian erythroblastosis virus (AEV), and all other retroviridae, including lentiviruses are included.

A detailed list of retroviruses can be found in Coffin et al. ("Retroviruses", 1997, Cold Spring Harbor Laboratory Press, JM Coffin, SM Hughes, HE Varmus, 758-763).

Lentiviruses also belong to the Retroviridae family, but lentiviruses can infect both dividing and non-dividing cells (Lewis et al. (1992) EMBO J. 3053-3058).

The lentivirus group can be classified into “primates” and “non-primates”. Examples of primate lentiviruses include human immunodeficiency virus (HIV), human acquired immunodeficiency syndrome (AIDS) virulence factors, and simian immunodeficiency virus (SIV). The non-primate lentivirus group has been described in the prototype “late-onset virus” visna / maedivirus (VMV), and related goat arthritis encephalitis virus (CAEV), equine infectious anemia virus (EIAV), and more recently Feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV) are included.

Details regarding the genomic structure of some lentiviruses can be found in the art. As an example, HIV and EIAV can be found in the NCBI Genbank database (ie, genome accession numbers AF033819 and AF033820, respectively). Details of HIV variants are also available at http: // hiv-web. lanl. gov. Can be found in Details of the EIAV mutants can be found at http: // www. ncbi. nlm. nih. gov. Can be found in

During the course of infection, retroviruses first attach to specific cell surface receptors. Upon entry into a susceptible host cell, the retroviral RNA genome is then copied into DNA by a virus-encoded reverse transcriptase that is retained within the parent virus. This DNA is transported to the host cell nucleus where it subsequently integrates into the host genome. This step is typically called a provirus. This provirus is stable in the host chromosome during cell division and is transcribed like other cellular genes. Proviruses also encode proteins and other factors necessary to make the virus and can leave the cell by a process sometimes referred to as “budding”.

Each retroviral genome contains genes called gag, pol, env that code for virion proteins and enzymes. These genes are flanked by regions called long terminal repeats (LTR). The LTR is involved in proviral integration and transcription. The LTR further acts as an enhancer / promoter sequence. In other words, the LTR can control the expression of viral genes. Retroviral RNA encapsidation occurs by a psi sequence located at the 5 'end of the viral genome.

The LTR itself is the same sequence that can be divided into three elements called U3, R, and U5. U3 is derived from a unique sequence at the 3 'end of the RNA. R is derived from a sequence that repeats at both ends of the RNA and U5 is derived from a unique sequence at the 5 'end of the RNA. The size of the three elements can vary considerably between the various retroviruses.

In the case of the viral genome, the transcription start site is the boundary between U3 and R of the left LTR, and the poly (A) addition (termination) site is the boundary between R and U5 of the right LTR. U3 contains most of the proviral transcriptional control elements, including a cellular transcriptional activation protein and, optionally, multiple enhancer sequences and promoters responsive to the viral transcriptional activation protein. Some retroviruses have one or more of any of the following genes, tat, rev, tax, rex, which code for proteins involved in the regulation of gene expression.

With respect to the structural genes, gag, pol, env itself, gag encodes the internal structural protein of the virus. The gag protein is processed by proteolysis into the mature proteins MA (substrate), CA (capsid), and NC (nucleocapsid). The pol gene encodes reverse transcriptase (RT), which contains DNA polymerase, related RNase H, and integrase (IN) and mediates genomic replication. The env gene encodes the surface (SU) glycoprotein and transmembrane (TM) protein of virions and forms a complex that specifically interacts with cell receptor proteins. This interaction ultimately results in infection by fusion of the viral and cell membranes.

Retroviruses can further contain “additional” genes encoding proteins in addition to gag, pol, env. Examples of additional genes include one or more of vif, vpr, vpx, vpu, tat, rev, and nef in HIV. EIAV has (among other things) the additional gene S2.

Proteins encoded by additional genes serve a variety of functions, some of which may be duplications of functions provided by cellular proteins. In EIAV, for example, tat acts as a transcriptional activator of the viral LTR. tat binds to a stable stem-loop RNA secondary structure called TAR. rev regulates and regulates viral gene expression by the rev response element (RRE). The mechanism of action of these two proteins is thought to be broadly similar to the similar mechanism of primate viruses. The function of S2 is unknown. Furthermore, the EIAV protein, Ttm, has been identified to be encoded by the first exon of tat spliced to the env coding sequence at the start of the transmembrane protein.

In one embodiment of the present invention, the lentiviral vector of the present invention is a recombinant lentiviral vector.

As used herein, the term “recombinant lentiviral vector” (RLV) allows the packaging of RNA genomes into viral particles that can infect and transduce target cells in the presence of packaging components. A vector with sufficient genetic information to make Target cell infection and transduction includes integration into the target cell genome and reverse transcription. RLV carries non-viral coding sequences that are delivered to target cells by the vector. RLV cannot produce infectious retroviral particles in the final target cell by independent replication. Usually, RLV lacks functional gag-pol and / or env genes and / or other genes necessary for replication. The vector of the present invention can be configured as a split-intron vector. Examples of split intron vectors are described in WO99 / 15683.

Preferably, the recombinant lentiviral vector (RLV) of the present invention has a minimal viral genome.

As used herein, the term “minimal viral genome” refers to non-essential elements to provide the required functionality to infect, transduce, and deliver a nucleotide sequence of interest to a target host cell. It means a viral vector that has been manipulated to remove and retain the essential elements. Further details regarding this strategy can be found in the applicant's WO 98/17815.

Thus, the minimal lentiviral genome used in the present invention comprises (5 ') R-U5-1 or more first nucleotide sequences-(regulatory element-NOI) _n- U3-R (3'). However, the plasmid vectors used to produce the lentiviral genome in the host / packaging cell may further function in the lentiviral genome to direct the transcription of the genome in the host / packaging cell. Linked transcriptional regulatory control sequences are included. These regulatory sequences can be transcriptional retroviral sequences, i.e. natural sequences associated with the 5 'U3 region, or can be heterologous promoters such as another viral promoter, e.g. the CMV promoter. Some lentiviral genomes require additional sequences for efficient virus production. For example, in the case of HIV, preferably rev and RRE sequences are included. However, the rev and RRE requirements can be reduced or eliminated using the present invention. Optional requirements for rev and RRE can be further reduced or eliminated by codon optimization. Further details of this strategy can be found in Applicants' WO 01/79518. Further, the rev requirement can be further reduced or eliminated by the use of a vector having at least one of the following: The ATG motif in the gag packaging signal of the wild type viral vector is the ATTG motif; the distance between the R regions of the viral vector is substantially the same as the distance in the wild type viral vector; the 3′U3 region of the viral vector is MLV A nucleotide sequence operably linked to a viral LTR, said nucleotide sequence upstream of an internal promoter, said nucleotide sequence preferably comprising a polypeptide or fragment thereof. Code.

Other sequences that perform the same function as the rev / RRE system are also known. For example, functional analogs of the rev / RRE system have been found in the Mason-Pfizer monkey virus. This is known as CTE and contains the RRE-type sequence of the genome that is thought to interact with factors in infected cells. This cellular factor can be considered a rev analog. Thus, CTE can be used as an alternative to the rev / RRE system. Any other functional equivalent known or made available may be relevant to the present invention. For example, it is also known that the HTLV-I Rex protein can functionally replace the HIV-1 Rev protein. Furthermore, Rev and Rex are known to have a similar effect to IRE-BP.

In a preferred embodiment, the viral genome of the first aspect of the invention lacks the Rev response element (RRE).

In a preferred embodiment, the system used in the present invention is based on a so-called “minimal” system in which some or all of the additional genes have been removed.

In one embodiment of the invention, the lentiviral vector is a self-inactivating vector. In other words, the viral promoter is a self-inactivating LTR.

As an example, self-inactivating retroviral vectors have been constructed by deleting transcription enhancers, or enhancers and promoters, in the U3 region of the 3'LTR. After reverse transcription and integration of a series of vectors, these changes are copied into both the 5 ′ and 3 ′ LTRs to produce a transcriptionally inactive provirus (Yu et al., 1986, Proc Natl Acad Sci 83: 3194. ~ 3198, Dougherty and Temin, 1987, Proc Natl Acad Sci 84: 1197-1201, Hawley et al., 1987, Proc Natl Acad Sci 84: 2406-2410, Yee et al. However, any promoter within the LTR of such vectors is still transcriptionally active. This strategy has been used to eliminate the influence of viral LTR enhancers and promoters on the transcription of genes located within. Such effects include increased transcription (Jolly et al., 1983, Nucleic Acids Res 11: 1855-1872) or transcriptional repression (Emerman and Temin, 1984, Cell 39: 449-467). This strategy can also be used to eliminate downstream transcription from the 3'LTR into genomic DNA (Herman and Coffin, 1987, Science 236: 845-848). This is particularly important in human gene therapy where prevention of exogenous activation of endogenous oncogenes is very important.

In one embodiment of the invention, the lentiviral vector is derived from a non-primate lentivirus. The non-primate lentivirus can be any member of the lentiviridae family that does not naturally infect primates and can be feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), goat arthritis encephalitis virus (CAEV). ), Maedi visna virus (MVV), or equine infectious anemia virus (EIAV). Preferably, the lentivirus is EIAV. Equine infectious anemia virus infects all equines and causes plasma viremia and thrombocytopenia (Clabough et al., 1991, J Virol. 65: 6242-51). Viral replication is thought to be regulated by the maturation process of monocytes into macrophages.

EIAV has the simplest genomic structure of a lentivirus and is particularly preferred for use in the present invention. In addition to the gag, pol, and env genes, EIAV encodes three other genes, tat, rev, and S2. Tat acts as a transcriptional activator of the viral LTR (Derse and Newbold, 1993, Virology. 194: 530-6, Maury et al., 1994, Virology. 200: 632-42), Rev is the rev response element (RRE ) Regulates and regulates the expression of viral genes (Martarano et al., 1994, J Virol. 68: 3102-11). The mechanism of action of these two proteins is believed to be broadly similar to the similar mechanism of primate viruses (Martano et al., Supra). The function of S2 is unknown. Furthermore, the EIAV protein, Ttm, has been identified to be encoded by the first exon of tat spliced to the env coding sequence at the start of the transmembrane protein.

In Applicants' WO 99/32646, Applicants provide details of features that can be advantageously applied to the present invention. In particular, the non-primate lentiviral genome comprises (1) a deletion gag gene wherein a deletion in gag preferably removes one or more nucleotides downstream of about nucleotide 350 or 354 of the gag coding sequence; 2) preferably has a lack of one or more accessory genes from the non-primate lentiviral genome, and (3) preferably lacks the tat gene but between the 5 ′ LTR end and the gag ATG. It is understood that it includes a leader sequence and is a combination of (4) (1), (2), and (3). In a particularly preferred embodiment, the lentiviral vector comprises all the features of (1) (2) (3).

Lentiviruses such as non-primate vectors can also be used, and the vector comprises at least one of the following: The ATG motif in the gag packaging signal of the wild type lentiviral vector is the ATTG motif; the distance between the R regions of the lentiviral vector is substantially the same as the distance in the wild type lentiviral vector; A U3 region comprising the sequence of an MLV U3 region; a nucleotide sequence operably linked to a viral LTR, said nucleotide sequence being upstream of an internal promoter, said nucleotide sequence preferably comprising a polypeptide or Code that fragment.

It is understood that the present invention can be used to deliver a nucleotide of interest (NOI) to a target cell. In another preferred embodiment of the first aspect of the invention, one or more nucleotides of interest (NOI) are introduced into the vector at the cloning site. Preferably, the NOI is a therapeutic gene, such therapeutic gene can be expressed from a promoter located in the retroviral LTR, or can be expressed from an internal promoter introduced at the cloning site. In a preferred embodiment, the NOI is introduced downstream of the internal regulatory element.

Delivery Systems Retroviral vector systems have been presented as delivery systems specifically for moving NOI to one or more sites of interest. This migration can occur in vitro, ex vivo, in vivo, or a combination thereof. Retroviral vector systems have been developed to study various aspects of the retroviral life cycle, including receptor usage, reverse transcription, and RNA packaging (Miller, 1992, Curr Top Microbiol Immunol 158: 1). ~ 24)).

Recombinant retroviral vector particles can transduce recipient cells with NOI. Within the cell, the RNA genome of the vector particle is reverse transcribed into DNA and incorporated into the recipient cell's DNA.

As used herein, the term “vector genome” refers to both the RNA construct present in the retroviral vector particle and the integrated DNA construct. The term further encompasses individual or isolated DNA constructs that can encode such RNA genomes. The retrovirus or lentiviral genome should contain at least one component derivable from the retrovirus or lentivirus. The term “inducible” does not necessarily have to be obtained from a virus such as a lentivirus, but is used in the usual sense to mean a nucleotide sequence or portion thereof that can be derived therefrom. As an example, the sequence can be prepared synthetically or by using recombinant DNA techniques. Preferably, the genome comprises a psi region (or similar component that can cause encapsidation).

The viral vector genome is preferably a “replication defect”, which allows us to enable independent replication of this genome alone to produce infectious viral particles in recipient cells. It means not containing enough genetic information. In preferred embodiments, the genome lacks a functional env, gag, or pol gene. In a highly preferred embodiment, the genome lacks the env, gag, and pol genes.

The viral vector genome can include some or all of the long terminal repeats (LTRs). Preferably, the genome comprises at least a portion of the LTR, or similar sequences that can mediate proviral integration and transcription. This sequence may further comprise an enhancer-promoter sequence or act as an enhancer-promoter sequence.

The viral vector genome of the first aspect of the present invention can be provided as a kit of parts. For example, the kit comprises (i) one or more plasmids containing NOI and internal regulatory element sequences, and (ii) a retro having appropriate restriction enzyme recognition sites for cloning the NOI and regulatory elements into the viral genome. Viral genome constructs can be included.

A retrovirus carrying a defective retroviral genome carrying a therapeutic gene by individual expression of components required to produce retroviral vector particles in separate DNA sequences co-introduced into the same cell It is known that particles are produced. This cell is called a producer cell (see below).

There are two general procedures for producing producer cells. In one procedure, sequences encoding retroviral Gag, Pol, and Env proteins are introduced into the cell and stably integrated into the cell genome. A stable cell line is produced, which is called a packaging cell line. Packaging cell lines produce the proteins required for retroviral RNA packaging, but are unable to cause encapsidation due to deletion of the psi region. However, when the vector genome (having the psi region) according to the first aspect of the invention is introduced into a packaging cell line, the helper protein packages the psi positive recombinant vector RNA to produce a recombinant virus strain. can do. This can be used to transduce NOI into recipient cells. Recombinant viruses whose genome lacks all the genes necessary for the production of viral proteins can infect only once but cannot propagate. Thus, NOI is introduced into the host cell genome without producing potentially harmful retroviruses. An overview of available packaging systems is described in “Retroviruses” (1997, Cold Spring Harbor Laboratory Press, JM Coffin, SM Hughes, HE Varmus, page 449).

The invention further provides a packaging cell line comprising the viral vector genome of the first aspect of the invention. For example, a packaging cell line can transduce a viral vector system containing the genome, or can be transfected with a plasmid carrying a DNA construct capable of encoding the RNA genome. The present invention further provides retroviral (or lentiviral) vector particles produced by such cells.

The second approach is to transiently pass a defective retroviral genome containing three different DNA sequences necessary to produce a retroviral vector particle: an env coding sequence, a gag-pol coding sequence, and one or more NOIs. This procedure is called transient triple transfection (Landau and Littman, 1992, Pear et al., 1993). The triple transfection method has been optimized (Soneoka et al., 1995, Finer et al., 1994). WO 94/29438 describes the production of producer cells in vitro using this multiplex DNA transient transfection method.

The components of the viral system that are required to complement the vector genome can be present in one or more “producer plasmids” for transfecting cells.

The present invention further includes
(I) a viral genome according to the first aspect of the invention,
(Ii) nucleotide sequences encoding lentiviral gag and pol proteins,
(Iii) Provide a vector system comprising a nucleotide sequence encoding other essential viral packaging components not encoded by the nucleotide sequence of ii). In a preferred embodiment, the nucleotide sequence of (iii) can encode an env protein. The invention further provides cells transfected with such a vector system and retroviral vector particles produced by such cells. Preferably, the gag-pol sequence is codon optimized for use in certain producer cells (see below).

The env protein encoded by nucleotide sequence iii) can be a homologous retroviral or lentiviral env protein. Alternatively, it can be a heterologous env, or an env from a non-retrovirus or lentivirus (see “Pseudotyping” below).

The term “viral vector system” is generally used to mean a kit of parts that can be used in combination with other components necessary for virus particle production to produce virus particles in a host cell. . For example, the retroviral vector genome may lack one or more genes necessary for viral replication. This can be combined, for example, in a kit with one or more additional complementary nucleotide sequences of one or more producer plasmids. Cotransfection of the genomic producer plasmid with the components provides the necessary components for the production of infectious viral particles.

Alternatively, the supplemental nucleotide sequence can be stably present in the packaging cell line included in the kit.

The present invention further relates to a retroviral vector system capable of delivering an RNA genome to a recipient cell, the genome being longer than the retroviral wild-type genome. For example, the vector system can be an EIAV vector system.

Preferably, the vector-based RNA genome has up to 5%, more preferably up to 10% more bases than the wild-type genome. Preferably, the RNA genome is about 10% longer than the wild type genome. For example, wild type EIAV contains an approximately 8 kb RNA genome. The EIAV vector system of the present invention has an RNA genome of up to 8.8 kb (preferably about 8.8 kb).

Preferably, the retroviral vector system of the present invention is a self-inactivating (SIN) vector system.

As an example, self-inactivating retroviral vector systems have been constructed by deleting transcription enhancers or enhancers and promoters in the U3 region of the 3'LTR. After a series of vector reverse transcription and integration, these changes are copied into both the 5 'and 3' LTRs, producing a transcriptionally inactive provirus. However, any promoter within the LTR of such vectors is still transcriptionally active. This strategy has been used to eliminate the influence of viral LTR enhancers and promoters on the transcription of genes located within. Such effects include increased transcription or suppressed transcription. This strategy can also be used to eliminate downstream transcription from the 3 'LTR into genomic DNA. This is particularly important in human gene therapy where prevention of exogenous activation of endogenous oncogenes is important.

Preferably, a recombinase assisting mechanism that promotes production of high titer-regulating lentiviral vectors from the producer cells of the present invention is used.

As used herein, the term “recombinase accessory system” includes, but is not limited to, a recombination event between the Cre recombinase / loxP recognition site of bacteriophage P1, or the 34 bp FLP recognition target (FRT). Which catalyzes S. cerevisiae site-specific FLP recombinases are used.

S. catalyzes recombination events between 34 bp FLP recognition targets (FRT). The site-specific FLP recombinase of C. cerevisiae has been constructed into a DNA construct to produce high levels of producer cell lines using recombinase-assisted recombination events (Karreman et al. (1996) NAR 24: 1616-1624). A similar system has been developed using the loxP recognition site of Cre recombinase / bacteriophage P1 (Vanin et al. (1997) J. Virol 71: 7820-7826). This was organized into the lentiviral genome so that a high titer lentiviral producer cell line was produced.

By using a producer / packaging cell line, for example, a large amount of retroviral vector particles for subsequent transduction of a site of interest (such as adult brain tissue) can be propagated and isolated (eg, of the appropriate titer). Retroviral vector particles can be prepared). Producer cell lines are usually advantageous for large-scale production or vector particles.

Transient transfection has many advantages over the packaging cell method. In this regard, transient transfection avoids the length of time required to generate a stable vector-producing cell line and is used when the vector genome or retroviral packaging components are toxic to the cell. It is done. If the vector genome encodes a toxic gene or a gene that interferes with host cell replication, such as a cell cycle inhibitor or a gene that induces apoptosis, it may be difficult to generate a stable vector-producing cell line. However, transient transfection can be used to produce vectors prior to cell death. In addition, cell lines have been developed that use transient infection to produce vector titer levels that correspond to those obtained from stable vector producing cell lines (Pear et al., 1993, PNAS 90: 8392-8396).

The producer cell / packaging cell line can be any suitable cell type. Producer cells are generally mammalian cells, but can be, for example, insect cells.

As used herein, the term “producer cell” or “vector producing cell” refers to a cell that contains all the elements necessary for the production of retroviral vector particles.

Preferably, the producer cell can be obtained from a stable producer cell line.

Preferably, the producer cell can be obtained from a stable induced producer cell line.

Preferably, the producer cell can be obtained from an induced producer cell line.

As used herein, the term “induced producer cell line” is a transduced producer cell line that has been screened and selected for high expression of a marker gene. Such cell lines maintain high levels of expression from the retroviral genome. The term “derived producer cell line” is used interchangeably with the terms “stable induced producer cell line” and “stable producer cell line”.

Preferably, the induced producer cell line includes, but is not limited to, retroviral and / or lentiviral producer cells.

Preferably, the induced producer cell line is an HIV or EIAV producer cell line, more preferably an EIAV producer cell line.

Preferably, the envelope protein sequence and nucleocapsid sequence are all stably incorporated into the producer and / or packaging cell. However, one or more of these sequences can also exist in an episomal form and gene expression can arise from the episome.

As used herein, the term “packaging cell” refers to a cell that contains the elements necessary for the production of an infectious recombinant virus that is deleted in the RNA genome. Typically, such packaging cells contain one or more producer plasmids that can express viral structural proteins (such as codon optimized gag-pol and env) but do not contain packaging signals.

The term “packaging signal”, synonymously “packaging sequence” or “psi”, is used in reference to non-coding, cis-acting sequences required for encapsidation of retroviral RNA strands during virion formation. . In HIV-1, this sequence maps to a locus that extends from upstream of the major splice donor site (SD) to at least the gag start codon.

A packaging cell line suitable for use with the vector constructs described above can be readily prepared (see also WO 92/05266) and utilized to create a producer cell line for producing retroviral vector particles. As already mentioned, an overview of available packaging systems is described in “Retroviruses” (above).

As also mentioned above, simple packaging cell lines containing proviruses in which the packaging signal has been deleted have been found to result in rapid production of undesired replicable viruses by recombination. To improve safety, a second generation cell line was produced in which the proviral 3 'LTR was deleted. In such cells, two recombinations are required to produce wild type virus. Further improvements include the introduction of gag-pol and env genes in individual constructs, so-called third generation packaging cell lines. These constructs are introduced sequentially to prevent recombination during transfection.

Preferably, the packaging cell line is a second generation packaging cell line.

Preferably, the packaging cell line is a third generation packaging cell line.

In these split constructs, third generation cell lines, further recombination reduction can be achieved by changing codons. This technique, based on the redundancy of the genetic code, aims to reduce the homology between individual constructs, eg between overlapping regions of the gag-pol and env open reading frames.

The packaging cell line is useful for providing the gene products necessary for encapsidation and the provision of membrane proteins to produce high titer vector particles. Packaging cells can be cultured in vitro, such as in tissue culture cell lines. Suitable cell lines include, but are not limited to, mammalian cells such as mouse fibroblast derived cell lines or human cell lines. Preferably, the packaging cell line is a human cell line, such as HEK293, 293-T, TE671, HT1080 and the like.

Alternatively, the packaging cells can be cells derived from the individual to be treated, such as monocytes, macrophages, blood cells, or fibroblasts. The cells can be isolated from the individual and administered ex vivo with packaging and vector components, followed by re-administration of autologous packaging cells.

More particularly, the packaging cells can be in vivo packaging cells in the body of the individual being treated, or can be cells cultured in vitro, such as a tissue culture cell line. Suitable cell lines include mammalian cells such as mouse fibroblast derived cell lines or human cell lines. Preferably, the packaging cell line is a human cell line, such as the 293 cell line, HEK293, 293-T, TE671, HT1080 and the like.

Alternatively, the packaging cells can be cells derived from the individual to be treated, such as monocytes, macrophages, stem cells, blood cells, or fibroblasts. The cells can be isolated from the individual and administered ex vivo with packaging and vector components, followed by re-administration of autologous packaging cells. Alternatively, packaging and vector components can be administered to the packaging cells in vivo. Methods for introducing lentiviral packaging and vector components into cells of an individual are known in the art. For example, one approach is to simultaneously transform various DNA sequences necessary to produce lentiviral vector particles, such as env coding sequences, gag-pol coding sequences, and defective lentiviral genomes by transient triple transfection. (Landau and Littman, 1992, J. Virol. 66, 5110, Sonoka et al., 1995, Nucleic Acids Res 23: 628-633).

In one embodiment, the vector structure of the present invention uses as its production system three transcription units that express the genome, the gag-pol component, and the envelope. The envelope expression cassette can include one of several envelopes such as VSV-G, or various mouse retroviral envelopes such as 4070A.

As usual, these three cassettes are expressed from three plasmids transiently transfected into an appropriate cell line, such as 293T, or a copy integrated into a stable producer cell line. Another approach is to use another virus, such as baculovirus or adenovirus, as the expression system for the three cassettes. Both of these are nuclear expression systems. To date, the use of poxviruses to express all components of the lentiviral vector system has not been described. In particular, given the unusual codon usage of lentiviruses and the requirements of RNA manipulation systems such as the rev / RRE system, the integration of all three cassettes and subsequent expression in the vector expressed in the cytoplasm rather than the nucleus It is not clear if it is feasible. To date, it remains possible that key nuclear factors and nuclear RNA engineering pathways are required for expression of vector components and function in gene delivery vehicles. Here we describe such a system and show that lentiviral components can be made in the cytoplasm and assemble them into a functional gene delivery system. The advantages of this system are the ability to easily manipulate poxviruses, high expression levels, and the ability to retain introns in the vector genome.

The lentiviral vector particles according to the present invention can further transduce cells that divide slowly and cannot be efficiently transduced by non-lentiviruses such as MLV. Slowly dividing cells, including certain tumor cells, divide once every about 3 to 4 days. Tumors contain cells that divide rapidly, but some tumor cells, especially those in the center of the tumor, have a low frequency of division. Alternatively, the target cell can be a cell in the central part of the mass, or a stem cell such as a hematopoietic stem cell, or a growth arrested cell that can undergo cell division such as a CD34 positive cell. As a further alternative, the target cells can be precursors of differentiated cells such as monocyte precursors, CD33 positive cells, or bone marrow precursors. As a further alternative, the target cells can be differentiated cells such as neurons, astrocytes, glial cells, microglia cells, macrophages, monocytes, epithelial cells, endothelial cells, or hepatocytes. Target cells can be transduced in vitro after isolation from a human individual or can be transduced directly in vivo.

In both experimental and practical applications, the use of high titer virus preparations is highly desirable. Techniques for increasing virus titer include the use of the psi and packaging signals described above, and virus stock enrichment.

As used herein, the term “high titer” refers to an effective amount of a retroviral vector or particle capable of transducing a target site such as a cell.

As used herein, the term “effective amount” means an amount of a regulatory retroviral or lentiviral vector or vector particle sufficient to induce expression of NOI at a target site.

Producer / packaging cell high titer virus preparations are typically on the order of 10 ⁵ to 10 ⁷ retroviral particles per ml. For transduction in tissues such as the brain, very small amounts need to be used, so that the virus preparation is concentrated by ultracentrifugation. The resulting preparation is at least 10 ⁸ t. u. / Ml, preferably 10 ⁸ to 10 ⁹ t. u. / Ml, more preferably at least 10 ⁹ t. u. (The titer is expressed as the titer of the standard D17 cell line, expressed in transducing units per ml (tu / ml)).

The NOI can be operably linked to one or more promoter / enhancer elements. The transcription of one or more NOIs can be under the control of the viral LTR, or the promoter / enhancer element can be incorporated using a transgene. Preferably, the promoter is a strong promoter such as CMV. The promoter can be a regulatable promoter. The promoter can be tissue specific.

The presence of a sequence called central polyprint lact (cPPT) may improve efficient gene delivery to non-dividing cells. This cis-acting element is located, for example, in the EIAV polymerase coding region element. Preferably, the genome of the present invention comprises a cPPT sequence.

Preferably, the viral genome includes post-translational regulatory elements. For example, the genome can include elements such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).

Additionally or alternatively, the viral genome can include a translation enhancer.

In the design of pseudotyping retroviral vector systems, it is desirable to create particles with different target cell specificities for the native virus so that genetic material can be delivered to an expanded or modified range of cell types. One way to achieve this is by creating a viral envelope protein to alter its specificity. Another approach is to introduce a heterologous envelope protein into the vector particle to replace or add to the native envelope protein of the virus.

The term pseudotyping means incorporating, replacing, or replacing all or part of the env gene of a viral genome with a heterologous env gene such as, for example, an env gene of another virus. Pseudotyping is not a new phenomenon, it is WO99 / 61639, WO-A-98 / 05759, WO-A-98 / 05754, WO-A-97 / 17457, WO-A-96 / 09400, WO-A-91. Examples can be found in / 00047 and Mebation et al., 1997, Cell 90, 841-847.

In a preferred embodiment of the invention, the vector system is pseudotyped with a gene encoding at least part of the rabies G protein. In another preferred embodiment of the invention, the vector system is pseudotyped with a gene encoding at least part of the VSV-G protein.

More specifically, the term “lentiviral particle” refers to a packaged retroviral vector that is preferably capable of binding and entering a target cell. As already discussed for vectors, the components of the particles can be modified for wild-type retroviruses. For example, the Env protein of the particle's protein coat can be genetically modified to alter the targeting specificity or to obtain some other desired function.

Preferably, the viral vector primarily transduces one or more cell types.

More preferably, the viral vector is a targeting vector, i.e., has a modified tissue affinity compared to the native virus such that the vector is targeted to specific cells. In the case of lentiviral vectors, this can be achieved by modification of the Env protein. The Env protein of the retroviral secondary vector should be a non-toxic envelope or an envelope that can be produced in a non-toxic amount in the primary target cell, such as an MMLV amphoteric envelope or a modified amphoteric envelope. The safety feature in such cases is preferably a lack of region or sequence homology between the lentiviral components.

Preferably, the envelope is one that allows transduction of human cells. Examples of suitable env genes include, but are not limited to, VS114 feline leukemia virus env or hemagglutinin (HA) from VS114 feline leukemia virus env, such as VSV-G, MLV amphoteric env, eg 4070A env. ) Is included. The Env protein may be capable of binding to a limited number of human cell type receptors and may be an engineered envelope containing a targeting moiety. The env and gag-pol coding sequences are transcribed from a promoter and optionally an enhancer active in a selected packaging cell line, the transcription unit being terminated by a polyadenylation signal. For example, if the packaging cell is a human cell, a suitable promoter-enhancer combination is derived from the human cytomegalovirus major immediate early (hCMV-MIE) gene and the polyadenylation signal of the SV40 virus can be used. . Other suitable promoters and polyadenylation signals are known in the art.

Open reading frame (ORF)
The first nucleic acid sequence is a sequence that can increase the level of genomic RNA available for packaging in the absence of Rev, for example, as compared to the absence of the first nucleic acid sequence. Preferably, the first nucleic acid sequence is an open reading frame (ORF) or fragment thereof that increases the level of genomic RNA available for packaging in the absence of Rev. By ORF, we will include a series of codon triplets that include a 5 ′ start codon (which can be a Kozak sequence) leading to a stop codon, representing a putative or known gene. However, any nucleic acid sequence that increases the level of genomic RNA for packaging can be used.

Thus, the present invention relates to a multicistronic vector genome that preferably has an ORF or partial ORF that is operably linked to a viral LTR and is downstream and upstream of a regulatory element such as an internal promoter or IRES.

When the vector genome is used with packaging components in the absence of Rev, such as the use of a codon optimized gag-pol gene, producing a complete minimal vector without accessory virus genes in the producer or target cell. it can.

Without wishing to be bound by any theory, Applicants have recognized in the system of the present invention that the transcription of the first nucleic acid sequence is recognized by the cell as being a “true” mRNA, and thus from the nucleus. Considered worthy of export. In the absence of such sequences, the mRNA is believed to be degraded in the nucleus, eg, by “nonsense-mediated degradation”, before being transported to the cytoplasm and subsequently packaged. The accessory protein Rev is believed to allow for the avoidance of this monitoring system and thus maintains the viral titer at which the vector genome is degraded in other situations. The present invention provides an alternative and safer method of maintaining virus titer.

Note that the genome in which a portion of the ORF has been reversed resulted in a different Rev dependence of the parent and derived genomes. Without wishing to be bound by any theory, Applicants believe that this is due to the presence of the ORF in the parent (Rev independent) genome, which was disrupted in the derived (Rev dependent) genome. Think. Therefore, the ORF should be in the correct direction, not the reverse direction.

Effectively, the ORF can be between about 0.6 and 4 kb, for example between about 0.65 or 0.7 and 3.1 or 3.0 kb.

In one embodiment, the first nucleic acid sequence is operably linked to the viral LTR, but can be operably linked to other regulatory elements as well.

Preferably, the first nucleic acid sequence can be a selection gene, also called a selectable marker. Typical selection genes confer resistance to antibiotics and other toxins such as ampicillin, neomycin, methotrexate, or tetracycline, supplement auxotrophic deficiencies, or supply key nutrients that are not available from complex media Encodes the protein to be Further details regarding suitable first nucleic acid sequences are described in the Reporter Gene section below.

Further, the first nucleic acid sequence can be the nucleotide of interest (NOI), and further details regarding NOI are described below, but generally, in order to obtain a good level of expression, the NOI is a promoter. Alternatively, it is preferably connected in a functional manner to an internal control element such as an IRES. However, if a low level of expression is desired, for example if the NOI is cytotoxic, the NOI may be operably linked to the LTR. In this embodiment, a nucleic acid sequence such as a marker gene can be placed downstream of the internal regulatory element.

In other preferred embodiments of the tetracycline-regulated expression system, the first nucleic acid sequence is a Tet repressor gene optionally linked to a nuclear localization signal (NLS). Without wishing to be bound by any theory, Applicants believe that the addition of NLS to the carboxy terminus is the concentration of TetR in the nucleus since the amino terminus of TetR is important for the binding of the tetracycline operator (tetO). To increase. Thus, in this embodiment, at least one of the downstream NOIs will be tetO. In a preferred embodiment, at least a secondary copy of the Tet repressor gene is present downstream of the IRES or internal promoter.

In other preferred embodiments, the Tet repressor gene is codon optimized for expression in a mammalian system. Further details of codon optimization are described below.

The present invention therefore relates to an improved TetR gene sequence for tetracycline-regulated expression in mammalian cells obtained by codon optimization. Regulated tetracycline expression systems have broad utility in many applications where inducible gene expression is advantageous or essential. Improvements in gene expression that may be due to codon-optimized effector proteins may significantly increase the efficiency of the system by promoting faster TetR-mediated expression.

The present invention further relates to modifying the 5 'sequence of the TetR gene so that the initial AUG is in a more favorable context for expression, thereby improving fidelity of initiation by eukaryotic ribosomes. In one embodiment, the 5 'sequence is 5' gccGCCCACAUGG3 '(G is in the +4 position, requiring a serine codon change or insertion of additional amino acid residues).

The invention further relates to NLS uptake that increases the local concentration of TetR in the nucleus.

Codon optimization Codon optimization has been previously described in WO 99/41397. Different cells use different specific codons. This codon bias corresponds to the relative abundance of a particular tRNA in the cell type. Expression can be increased by changing codons in the sequence to match the relative abundance of the corresponding tRNA. Similarly, expression can be reduced by deliberately selecting codons for which the corresponding tRNA is known to be rare in a particular cell type. Thus, further translational control can be obtained.

Many viruses, including HIV and other lentiviruses, use a large number of rare codons, and by changing them to correspond to commonly used mammalian codons, expression of packaging components in mammalian producer cells An increase in can be achieved. Codon usage tables are known in the art for mammalian cells, as well as various other organisms.

Codon optimization has several other advantages. The sequence modification eliminates RNA labile sequences (INS) from the nucleotide sequence encoding the viral particle packaging component required for viral particle assembly in producer / packaging cells. At the same time, the amino acid coding sequence of the packaging component is maintained, so that the viral component encoded by that sequence is kept identical or at least sufficiently similar so that the function of the packaging component is not impaired. . Codon optimization further overcomes the Rev / RRE requirement for export and imparts Rev independence to the optimized sequence. Codon optimization further reduces homologous recombination between different constructs within the vector system (eg, between overlapping regions of the gag-pol and env open reading frames). Thus, the overall effect of codon optimization is a significant increase in virus titer and improved safety.

In one embodiment, only codons associated with INS are optimized. However, in a much more preferred and practical embodiment, the sequence is codon-optimized in its entirety, except for sequences that include frameshift sites.

The gag-pol gene contains two overlapping reading frames encoding the gag and pol proteins, respectively. The expression of both proteins depends on the frameshift during translation. This frameshift occurs as a result of a “shift” of the ribosome during translation. This shift is believed to be due at least in part to the secondary structure of ribosome-stopped RNA. Such secondary structure exists downstream of the frameshift site of the gag-pol gene. In the case of HIV, the overlapping region extends from nucleotide 1222 downstream of gag (nucleotide 1 is A of gag ATG) to the end of gag (nucleotide 1503). As a result, the 281 bp fragment spanning the frameshift site and the overlapping site of the two reading frames is preferably not codon optimized. By retaining this fragment, more efficient expression of the gag-pol protein is possible.

In the case of EIAV, the beginning of the overlap is considered to be nucleotide 1262 (nucleotide 1 is A of gag ATG). The end of the overlap is at 1491 bp. The wild type sequence was retained from nucleotides 1156 to 1465 to ensure conservation of the frameshift site and gag-pol overlap.

For example, optimal codon usage can be derived to match convenient restriction sites and conservative amino acid changes can be introduced into the gag-pol protein.

In a highly preferred embodiment, codon optimization was based on easily expressed mammalian genes. The third base, optionally the second and third bases can be changed.

It is understood that due to the degeneracy nature of the genetic code, a number of gag-pol sequences can be obtained by engineers. In addition, a number of retroviral variants have been described that can be used as starting points for codon-optimized gag-pol sequence production. The lentiviral genome can be very variable. For example, there are many subspecies of HIV-1 that are still functional. The same applies to EIAV. These mutants can be used to improve certain parts of the transduction method. Examples of HIV-1 mutants can be found at http: // hiv-web. lanl. gov. Can be found in Details of the EIAV clone can be found in the NCBI database http: // www. ncbi. nlm. nih. gov. Can be found in

Codon optimized gag-pol and TetR sequence strategies can be used for any retrovirus. This applies to all lentiviruses including EIAV, FIV, BIV, CAEV, VMR, SIV, HIV-1 and HIV-2. Furthermore, this method can also be used to increase the expression of HTLV-1, HTLV-2, HFV, HSRV, and human endogenous retrovirus (HERV), MLV, and other retrovirus genes.

Codon optimization can make gag-pol expression Rev independent. However, it would be necessary to make the viral vector production system completely Rev / RRE independent to allow the use of anti-rev or RRE factors in retroviral vectors. Therefore, this genome needs to be further modified. This is achieved by optimizing the vector genomic components according to the present invention. Advantageously, these modifications further result in the production of a safer system in the absence of any additional protein in both producer and transduced cells.

As described above, the packaging components of retroviral vectors include the expression products of gag, pol, and env genes. Furthermore, efficient packaging relies on a short sequence of four stem loops followed by a partial sequence of gag and env (“packaging signal”). Thus, by including the deleted gag sequence in the retroviral vector genome (in addition to the complete gag sequence of the packaging construct), the vector titer is optimized. To date, efficient packaging requires about 255 to 360 nucleotides of gag in a vector that still retains the env sequence, or about 40 nucleotides of gag in a particular combination of splice donor mutations, gag and env deletions. It has been reported. Surprisingly, it was found that deleting all but about 360 nucleotides of the N-terminus of gag increased the vector titer. Thus, preferably, the retroviral vector genome comprises a gag sequence comprising one or more deletions, more preferably the gag sequence comprises about 360 nucleotides derivable from the N-terminus.

The TetR sequence can be effectively codon optimized using the codons shown in FIG.

NOI
In the present invention, the term NOI (nucleotide sequence of interest) includes any suitable nucleotide sequence and does not necessarily have to be a fully natural DNA or RNA sequence. Thus, the NOI can be, for example, a synthetic RNA / DNA sequence, a recombinant RNA / DNA sequence (ie, a sequence prepared using recombinant DNA technology), a cDNA sequence, or a partial genomic DNA sequence, and combinations thereof including. This sequence need not be a coding region. If the sequence is a coding region, it need not be the entire coding region. Furthermore, the RNA / DNA sequence can be sense oriented or antisense oriented. Preferably, it is sense orientation. Preferably, this sequence comprises or is transcribed from cDNA.

The NOI, also referred to as “heterologous sequence”, “heterologous gene”, or “transgene” can be, for example, any one or more selection genes, marker genes, and therapeutic genes.

NOI can also be a candidate gene that may be important in the disease process. Therefore, the vector system of the present invention can be used for target confirmation, for example.

The NOI can have therapeutic or diagnostic applications. Suitable NOIs include but are not limited to enzymes, cytokines, chemokines, hormones, antibodies, antioxidant molecules, engineered immunoglobulin-like molecules, single chain antibodies, fusion proteins, immune costimulatory molecules, immune Regulatory molecules, antisense RNAs, transdominant negative mutants of target proteins, toxins, conditional toxins, antigens, tumor suppressor proteins and growth factors, membrane proteins, vasoactive proteins and peptides, antiviral proteins and ribozymes, and derivatives thereof Sequences encoding (such as related reporter groups) are included. The NOI can also encode a prodrug activating enzyme.

The NOI coding sequence can encode a fragment of the coding sequence.

The lentiviral vectors of the present invention can be used to deliver NOI such as prodrug activating enzymes to tumor sites to treat cancer. In either case, a prodrug suitable for treatment of an individual (such as a patient) is used in combination with a suitable prodrug activating enzyme. An appropriate prodrug is administered with the vector. Examples of prodrugs include phosphate etoposide (with alkaline phosphatase; Senter et al., 1988, Proc Natl Acad Sci 85: 4842-4846), 5-fluorocytosine (with cytosine deaminase. Mullen et al., 1994, Cancer Res 54: 1503. ˜1506), doxorubicin-Np-hydroxyphenoxyacetamide (with penicillin-V-amidase. Kerr et al., 1990, Cancer Immunol Immunoter 31: 202-206), para-N-bis (2-chloroethyl) aminobenzoyl glutamate ( With carboxypeptidase G2), cephalosporin nitrogen mustard carbamate (with β-lactamase), SR-4233 (P4 0 with reductase), ganciclovir (with HSV thymidine kinase, Borrelli et al., 1988, Proc Natl Acad Sci 85: 7572-7576), mustard prodrug, with nitroreductase (Friedlos et al., 1997, J Med Chem 40: 1270-1275). , And cyclophosphamide (with P450. Chen et al., 1996, Cancer Res 56: 1331-1340).

Preferably, NOI is useful for the treatment of neurodegenerative diseases.

More preferably, NOI is useful for the treatment of Parkinson's disease.

The NOI can encode an enzyme involved in dopamine synthesis. For example, the enzyme can be one of the following tyrosine hydroxylases, GTP-cyclohydrase I and / or the aromatic amino acid dopa decarboxylase. Sequences for all three genes are available. The accession numbers are X05290, U19523, and M76180, respectively.

Alternatively, the NOI can encode vesicular monoamine transporter 2 (VMAT2). In a preferred embodiment, the viral genome comprises a NOI that encodes the aromatic amino acid dopa decarboxylase and a NOI that encodes VMAT2. Such a genome can be used in the treatment of Parkinson's disease, particularly with peripheral administration of L-dopa.

Alternatively, the NOI can encode a factor that can block or inhibit denaturation of the substantia nigra system. An example of such a factor is a neurotrophic factor. For example, the NOI can encode glial cell line derived neurotrophic factor (GDNF), or brain derived neurotrophic factor (BDNF).

Alternatively, the NOI can encode a neuroprotective factor. In particular, the NOI can encode a molecule that prevents the death of TH positive neurons, or a molecule that promotes regeneration and functional recovery in the damaged nigrostriatal system.

The NOI can encode all or part of a protein of interest (“POI”), or a mutant, homologue, or variant thereof. For example, the NOI can encode a fragment of POI that can function in a manner similar to the wild-type protein in vivo.

In a highly preferred embodiment, one of the NOIs includes a truncated form of the TH gene that lacks the regulatory region. Such NOI avoids feedback suppression by dopamine which may limit full-length enzyme expression.

The term “mutant” includes POIs that contain one or more amino acid variations of the wild type sequence. For example, a mutant can include one or more amino acid additions, deletions, or substitutions. Mutants can occur naturally or can be made artificially (eg, by site-directed mutagenesis).

IRES
The viral genome of the first aspect of the invention includes one or more NOIs. Because multiple NOIs are expressed, there can be more than one transcription unit in the vector genome, one for each NOI. However, if the retroviral vector remains genetically simple, the retroviral vector acquires the highest titer and very strong gene expression properties (PCT / GB96 / 01230, Bowtel et al., 1988, J. MoI. Virol.62, 2464, Correll et al., 1994, Blood 84, 1812, Emerman and Temin, 1984, Cell 39, 459, Ghattas et al., 1991, Mol. 3519, Hatzoglou et al., 1991, J. Biol. Chem 266, 8416, Hatzoglou et al., 1988, J. Biol. Chem 263, 17798, Li et al., 1992, Hum. Ge. Ther.3, 381, McLachlin et al., 1993, Virol.195, 1, Overell et al., 1988, Mol. Cell Biol. 307, Xu et al., 1989, Virol. 171, 331, Yee et al., 1987, PNAS 84, 5197), so use IRES to initiate translation of the second (and subsequent) coding sequence in polycistronic messages. It is clear from the literature that it is preferred (Adam et al., 1991, J. Virol. 65, 4985).

Insertion of an IRES element into a retroviral vector is compatible with the retroviral replication cycle and allows expression of multiple coding regions from a single promoter (Adam et al. (Supra), Koo et al., 1992, Virology 186: 669). ~ 675, Chen et al., 1993, J. Virol. 67: 2142-2148). IRES elements were first found at the untranslated 5 'end of Picornavirus, which promotes cap-independent translation of viral proteins (Jang et al., 1990, Enzyme 44: 292-309). When located between RNA open reading frames, the IRES element allows efficient translation of the downstream open reading frame by facilitating ribosome entry in the IRES element, followed by downstream translation initiation.

A review on IRES has been presented by Mountford and Smith (TIG, May 1995, vol 11, No 5: 179-184). Encephalomyocarditis virus (EMCV) (Ghattas, IR, et al., Mol. Cell. Biol. 11: 5848-5859, 1991), BiP protein (Macejak and Sarnow, Nature 353: 91, 1991), Drosophila Antennapedia gene (Exons d and e) (Oh et al., Genes & Development 6: 1643-1653, 1992), and poliovirus (PV) (Pelletier and Sonenberg, Nature 334: 320-325, 1988, Mountford and Smith, TIG 11, 179-184) , See also 1985) several different IRES sequences are known, including sequences derived from them.

According to WO-A-97 / 14809, IRES sequences are typically found in the 5 'non-coding region of genes. In addition to those described in this document, the IRES sequence looks for gene sequences that affect expression, and then that sequence affects DNA (ie, acts as a promoter or enhancer) or only affects RNA. Can be found empirically by determining whether it acts as an IRES sequence.

IRES elements from PV, EMCV, and porcine varicella disease virus have been used in retroviral vectors so far (Coffin et al., Supra).

The term “IRES” includes any sequence or combination of sequences that acts as an IRES or that improves the function of an IRES.

The IRES can be virus derived (such as EMCV IRES or PV IRES) or cell derived.

In order for an IRES to be able to initiate translation of each NOI, the IRES should be located between the NOIs of the vector genome. In other words, there is always one less IRES sequence compared to NOI. For example, for a multicistronic sequence containing n NOIs, the genome can be as follows:
_{[(NOI 1-n-1} ) - (IRES 1-n-1)] - NOI n

For 2 and 3 cistronic sequences, the order can be as follows:
NOI ₁ -IRES ₁ -NOI ₂
NOI ₁ -IRES ₁ -NOI ₂ -IRES ₂ -NOI ₃

One or more NOIs may be operably linked to an internal promoter. The discussion on promoters is shown below. It is also possible to use a combination of promoter and IRES.

Transduced cells The invention further relates to cells transduced with a vector system comprising a viral genome according to the first aspect of the invention.

Transduction with the vector system of the present invention can confer or increase the ability of cells to produce catecholamines. For example, the ability of a cell to convert tyrosine to L-dopa and / or L-dopa to dopamine can be imparted or increased. Catecholamine release can be measured using techniques known in the art, such as an electrochemical detector connected to an analytical cell. In addition to catecholamine itself, byproducts associated with catecholamine release (DOPAC, specific degradation products of dopamine, etc.) can also be detected.

Cells can be transduced in vivo, in vitro, or ex vivo. For example, if the cells are cells from a mammalian subject, the cells can be removed from the subject and transduced in preparation for retransplantation into the subject (ex vivo transduction). Alternatively, cells can be transduced by direct gene transfer in vivo using the vector system of the present invention according to standard techniques (eg, by injection of a vector stock expressing NOI). If the cell is part of a cell line that is stable in culture (ie one that can survive in multiple subcultures and can be grown in vitro), it can be produced by standard techniques, eg on a virus containing a vector expressing NOI. Transduction can be done in vitro by exposing the cells to a cell.

The cell can be any cell that can be transduced. If the vector system can transduce non-dividing cells (eg, if it is a lentiviral system), the cells can be non-dividing cells such as neurons.

In a preferred embodiment, the transduced cells form part of a genetically modified neural cell line. Such cell lines can be transplanted into the brain, for example for the treatment of Parkinson's disease.

In other embodiments, the cell is a neural stem cell. Such cell lines can be transplanted into the brain, for example for the treatment of Parkinson's disease.

In other embodiments, the cells are cells of the striatum of interest, such as neurons or glial cells. Such cells can be converted, for example, to dopamine producer cells by direct gene transfer in vivo.

Cassettes The present invention further provides multicistronic cassettes comprising two or more NOIs and ORFs operably linked by regulatory elements such as IRES or internal promoters. This cassette can be used in a method of producing a vector genome in a producer cell.

The invention further provides an expression vector comprising such a cassette. Transfection of appropriate cells with such an expression vector results in cells expressing each POI encoded by the NOI of the cassette. The invention further provides such transfected cells.

Cloning of the cassette into an expression vector and transfection of the cell with the vector (to bring about expression of the cassette) can be performed by techniques well known in the art (Sambrook et al. (Molecular Cloning: A Laboratory Manual). , 2nd edition, Cold Spring Harbor Laboratory press, 1989) and other experimental documents).

Preferably, the cassette includes a promoter. In a highly preferred embodiment, the cassette is dicistronic or tricistronic and comprises the following elements:
LTR-ORF-promoter / IRES- (NOI ₁ )-(IRES ₁ ) -NOI ₂ )
LTR-ORF-promoter / IRES- (NOI ₁ )-(IRES ₁ )-(NOI ₂ )-(IRES ₂ )-(NOI ₃ )

In a particularly preferred embodiment, the cassette is dicistronic, a NOI encoding a tyrosine hydroxylase (or mutant, variant or homologue thereof), and GTP-cyclohydrolase I (or mutant thereof) NOI encoding the mutant or homolog).

In another particularly preferred embodiment, the cassette is tricistronic and encodes a tyrosine hydroxylase (or mutant, variant or homologue thereof) NOI, GTP-cyclohydrolase I (or mutant thereof). , Variants, or homologues) and NOIs encoding aromatic amino acid dopa decarboxylase (or mutants, variants, or homologues thereof).

Targeting moieties and signaling pathways Aspects of the invention allow identification of unidentified components of signaling pathways. Such components are referred to herein as “target moieties”. An identifying component or target portion of a signal transduction pathway may be useful as a drug target. The expression “signal transduction pathway” refers to any one or more events that result in or result from (and including) activation of a pathway. Preferably, by “signal transduction pathway” we refer to a signal transduction pathway comprising elements that interact genetically and / or molecularly. This signal transduction pathway includes signal transduction events that occur extracellularly or at the cell membrane, as well as signal transduction events that occur within the cell, eg, within the cytoplasm or inside the cell nucleus.

There are no particular restrictions on the signaling pathways to which the present invention can be applied, but phenotypic differences should be measurable.

Examples of pathways to which the present invention can be usefully applied include the Neu-Ras pathway associated with breast cancer, and the apoptotic pathway. Several other pathways have been implicated in breast cancer and other cancers, such as the ErbB receptor family, estrogen receptor, insulin-like growth factor (IGF), integrin receptor system. In prostate cancer, the androgen receptor (AR) signaling pathway is thought to play an important role in the early development as well as later progression of the disease.

A non-limiting list of disease states to which the present invention applies is provided below. For example, the present invention may be useful for Parkinson's disease and other neurological diseases.

In patients with sickle cell disease, for example, it may be advantageous to induce the expression of fetal hemoglobin. The promoter of this gene driving reporter expression can be used to identify a peptide of cDNA that induces fetal hemoglobin expression. However, if the expression of this gene is suppressed in adults, the use of an antisense or ribozyme library may be more appropriate. Similarly, in the case of Duchenne muscular dystrophy (DMD) due to mutational loss of the protein dystrophin, research has focused on up-regulation of the utrophin gene.

Modulating moiety A modulating moiety is a compound that can affect a signaling pathway by binding to a targeting moiety, ie, can up-regulate or down-regulate a signaling pathway.

As used herein, the term “modulate” refers to a change in biological activity of a signal transduction pathway. Thus, modulation of the pathway includes inhibition or down-regulation of signal transduction, for example by compounds that block at least some normal biological activity of the signal transduction pathway. Alternatively, the term “modulation” can refer to activation or upregulation of signal transduction, eg, by a compound that promotes or upregulates the normal biological activity of the signal transduction pathway, at least in part.

By modulation of signal transduction, the applicants
(A) (i) Dominant inhibition or inhibitor of suppressor, and (ii) Activation of signal transduction pathway by activator, and (b) (i) Dominant inhibition or inhibitor of activator, and (ii) ) Including blocking signal transduction pathways by inhibitors.

The modulating moiety is preferably selected from polypeptides and fragments thereof, linear peptides, cyclic peptides, nucleic acids encoding them, synthetic or natural compounds including low molecular weight organic or inorganic compounds, ribozymes, antisense molecules, and antibodies Is done.

Non-functional homologues of the polypeptides of the present invention may also be unable to perform the normal function of the protein, but may compete with wild-type proteins for binding to other components of the signal transduction pathway, so any It is understood that one can test for modulation of a given signaling pathway (eg, for inhibition of cell cycle progression). Alternatively, a non-functional homologue of a polypeptide of the invention may block protein function when bound to a signal transduction pathway component. Such non-functional homologues can include natural mutants and modified sequences or fragments thereof.

Alternatively, instead of directly hindering the binding of the components, the substance can suppress the bioavailable amount of the polypeptide of the invention. This can be due to, for example, inhibiting the expression of components at the level of transcription, transcription stability, translation or post-translational stability. Examples of such substances are antisense RNA or double stranded interfering RNA sequences that suppress the amount of mRNA biosynthesis.

Suitable candidate substances are peptides based on the sequence of the polypeptides described in the examples, in particular peptides with a size of about 5 to 30 or 10 to 25 amino acids, or one or more residues. Variants of such peptides are included. Peptides derived from a random sequence or a panel of peptides containing sequences that have been sequentially modified to provide the most diverse panel of peptides can be used.

Suitable candidate substances further include antibody products (eg, monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, and CDR grafted antibodies) specific for the polypeptides of the invention. In addition, combinatorial libraries, peptides and peptidomimetics, defined chemicals, oligonucleotides, natural product libraries can also be used in signal transduction pathway mechanisms, such as mitotic / meiotic apparatus (such as microtubules). Screening for activity as inhibitors of binding of the polypeptides of the invention to other components of the mechanism can be made. Candidate substances can be used, for example, for initial screening in batches containing 10 substances per reaction, and substances that show inhibition of those batches are tested individually.

Candidate substances are typically added at a final concentration of 1 to 1000 nmol / ml, more preferably 1 to 100 nmol / ml. In the case of antibodies, the final concentration used is typically 100 to 500 μg / ml, more preferably 200 to 300 μg / ml.

Candidate substances can be identified by in vitro assays before being tested on whole cells. Alternatively, a whole cell assay, preferably in the form of a fast throughput screen, can be used as a preliminary screen, followed by an in vitro assay to confirm its effect. Suitable in vitro assays that can be used include binding assays, in which the polypeptide is immobilized on a solid support, the candidate substance is non-immobilized, and the polypeptide and candidate substance bind to each other. And / or how much to combine. Alternatively, the candidate substance may be fixed and the polypeptide may be non-immobilized.

In one embodiment, a library of candidate modulating moieties is created or used.

Library As used herein, the term “library” includes, but is not limited to, an accumulation of nucleic acid fragments encoding a candidate modulating moiety (herein, a candidate modulating moiety). Called a library).

The libraries of the present invention include, but are not limited to, in vitro mutagenesis and / or recombination methods such as chemical treatment, oligonucleotide-mediated mutagenesis, PCR mutagenesis, DNA shuffling, random priming It can be produced by methods including recombination (RPR), restriction enzyme fragment induced template switching (REFITS), attachment extension process (StEP) and the like. The library of sequence variants can be random, semi-random, or a known sequence.

The nucleotide sequence screened by the present invention typically encodes a polypeptide having the activity of interest, or an RNA molecule having catalytic activity, such as a ribozyme. However, nucleotide sequences that may have activity as non-transcribed molecules, such as sequence motifs bound by DNA binding proteins, can also be screened.

Polypeptides having a target activity include antibodies, antigens, enzymes, ligands such as growth factors, receptors such as cell surface receptors, components of other cell signaling pathways, DNA-binding proteins such as DNA repair enzymes, Polymerases, recombinases, and transcription factors, and structural proteins are included. Polypeptides also include the fragments described above.

Polypeptides used in the screening methods of the present invention can include families of polypeptides having known or candidate nucleic acid binding motifs. These can include, for example, zinc finger proteins (see below). Molecules according to the present invention may further comprise a helix-turn-helix protein, homeodomain, leucine zipper protein, helix-loop-helix protein, or β-sheet motif that is well known to those skilled in the art. Can be included.

By way of example, a DNA binding protein can be a DNA binding portion of a protein, particularly a zinc finger transcription factor, Zif268, ATF family transcription factor, ATF1, ATF2, bZIP protein, CHOP, NF-κB, TATA binding protein (TBP), MDM, c-jun, elk, serum response factor (SRF), triple complex factor (TCF), etc., KRUPPEL, odd Skipped, even skipped, and other D.C. It can include a melanogaster transcription factor, a yeast transcription factor such as GCN4, a GAL family of galactose-inducible transcription factors, a bacterial transcription factor or repressor such as LacI ^q , or a fragment or derivative thereof. Derivatives are considered functionally and / or structurally related to the molecule from which they are derived, eg by at least 40% sequence homology, by those skilled in the art.

The polypeptide can be a non-randomized polypeptide, such as a “wild type” or allelic variant of a natural polypeptide, or can be a specific mutant, or a fully or partially randomized polypeptide For example, it can be a randomized library of DNA binding proteins as described herein, particularly where the DNA binding motif is randomized.

Preferably, amino acid residues known to be important for polypeptide function, such as amino acid residues known to be important for ligand binding or catalytic activity such as nucleic acid binding or antigen binding, etc. Has been. Detailed guidance of residues that can be randomized, such as zinc finger proteins and several other transcription factors, are given in WO 98/53057, WO 98/53060, WO 98/53058, WO 98/53059.

Randomization is achieved at the nucleotide level by any suitable means of mutagenesis. Mutagenesis can be performed, for example, by synthesizing novel genes encoding mutant polypeptides and expressing them to obtain a variety of different proteins. Alternatively, the existing gene itself can be mutated by site-directed or random mutagenesis to obtain the desired mutated gene.

Mutations can be made by any method known to those skilled in the art. However, site-directed mutagenesis of the nucleic acid sequence encoding the protein of interest is preferred. Several methods of site-directed mutagenesis are known in the art, from methods using single-stranded phages such as M13 to PCR-based techniques ("PCR Protocols: A guide to methods and applications", M A. Innis, DH Gelfund, JJ Sninsky, TJ White (eds.), Academic Press, New York, 1990). Preferably, a commercially available Altered Site II Mutagenesis System (Promega) can be used according to the manufacturer's instructions. Other techniques are described below.

A variety of nucleotide sequences encoding the polypeptide of interest can be cloned from genomic DNA or cDNA. For example, libraries generated by PCR amplification of antibody gene repertoires from immune or non-immune donors have been shown to be a very effective source of functional antibody fragments (Winter et al., 1994, Annu Rev. Immunol, 12, 433-55, Hoogenboom, 1997, Trends Biotechnol. 15, 62-70). A library of genes can also be generated, which can be all genes (see, eg, Palmley and Smith, 1988, Gene, 73, 305-18) or parts (eg, Lowman et al., 1991, Biochemistry, 30, 10832- 8), or a pool of genes (see eg Nissim et al., 1994, Embo J, 13, 692-8).

Various nucleotide sequences are described in E.I. including the use of bacterial “mutagenesis strains” such as E. coli mutD5 (Low et al., 1996), and the use of antibody hypermutation systems for B lymphocytes (Yelamos et al., 1995, Nature, 376, 225-9). It can also be made by introducing mutations into a nucleotide sequence, or pool of nucleotide sequences, randomly, by a variety of in vivo techniques. Random mutations include chemical mutagens and ionization or UV irradiation (see Friedberg et al., 1995, DNA repair and mutationage, ASM Press, Washington DC), or incorporation of mutagenic base analogs (Zaccolo et al. , 1996, J Mol Biol, 255, 589-603) can be introduced both in vivo and in vitro. “Random” mutations can also be introduced into genes in vitro during polymerization, for example using a mutated polymerase (Leung et al., 1989, Technique 1, 11-15).

For further diversification, see in vivo (see Kowalczykowski et al., 1994, Microbiol Rev, 58, 401-65) or in vitro (Stemmer, 1994a, Nature, 370, 389-91, Stemmer, 1994b, Proc Natl Acad Sc, 91, 10747-51) can be introduced using homologous recombination.

A particularly preferred library of nucleotide sequences for use in the present invention is a PCR assembled combinatorial library.

Theoretical studies suggest that the greater the number of genetic factor variants created, the greater the likelihood that a molecule with the desired properties will be created. Recently, it has been experimentally confirmed that a larger repertoire of phage-antibody actually results in more antibodies with better binding affinity than a small repertoire. Thus, large size libraries are desirable to ensure that rare variants can be produced and selected. Thus, a library of nucleotide sequences used in the present invention typically comprises at least 100, 500, 1000, or 10,000 different nucleotide sequences, preferably at least 10 ⁵ , 10 ⁶ , or 10 ⁷ different nucleotide sequences. .

The plurality of nucleotide sequences used in the screening method of the present invention can include RNA or DNA, but typically includes DNA. Where the nucleotide sequence encodes an RNA sequence such as mRNA or a catalytic ribozyme, the nucleotide sequence typically includes, in addition to the coding sequence, transcription regulatory sequences suitable for directing expression of the coding sequence in vitro. Provided as part of a nucleic acid vector. For example, a vector containing a T7 or T3 promoter upstream of the coding sequence can be used. Nucleotide sequences can be provided as linear or circular DNA. Where the coding sequence encodes a polypeptide, the coding sequence typically further comprises, in addition to the sequence encoding the mature protein, a sequence that directs translation of the coding sequence in an in vitro system.

Preferably, the library candidate modulating moiety comprises a group of ribozymes or antisense molecules.

Ribozymes are RNA molecules that can function to catalyze specific chemical reactions in cells without the essential involvement of proteins. For example, Group I ribozymes take the form of introns that can mediate self-excision from self-splicing precursor RNA. Other ribozymes are derived from self-cleaving RNA structures that are essential for the replication of viral RNA molecules. Like protein enzymes, ribozymes can be folded into secondary and tertiary structures that provide specific binding sites for cofactors such as substrates and metal ions. Examples of such structures include hammerheads, hairpins, or stem loops, and pseudoknot and hepatitis delta antigenomic ribozymes have been described.

Each individual ribozyme has a motif that recognizes and binds to the recognition site of the target RNA. This motif takes the form of one or more “binding arms” but generally takes the form of two binding arms. The binding arm of the hammerhead ribozyme is the flanking sequences Helix I and Helix III adjacent to Helix II. These can be of various lengths, usually 6 to 10 nucleotides each, but can be shorter or longer. The length of flanking sequences can affect the rate of cleavage. For example, it has been found that reducing the number of adjacent nucleotides from 20 to 12 can increase the turnover rate of ribozymes that cleave HIV sequences by a factor of 10 (Goodchild, JVK, 1991, Arch Biochem Biophys 284: 386-391). The catalytic motif of the ribozyme Helix II of the hammerhead ribozyme cleaves the target RNA at a site called a cleavage site. Whether a ribozyme cleaves any given RNA is determined by the presence or absence of a recognition site for a ribozyme containing the appropriate cleavage site.

Each type of ribozyme recognizes its own cleavage site. The hammerhead ribozyme cleavage site has a nucleotide base triplet GUX immediately upstream, where G is guanine, U is uracil, and X is any nucleotide base. The hairpin ribozyme has a BCUGNYR cleavage site, wherein B is any nucleotide base other than adenine, N is any nucleotide, Y is cytosine or thymine, and R is guanine or adenine. is there. At this cleavage site, cleavage by hairpin ribozyme occurs between G and N.

For further details on ribozymes, see “Molecular Biology and Biotechnology” (RA Meyers, 1995, VCH Publishers Inc, pages 831-8320), “Retroviruses” (JM Coffin et al., 1997, Cold Spring 68, Cold Spring Phr. And in the applicants WO 99/41397 and WO 00/75370.

Alternatively, instead of directly hindering the binding of the components, the substance can suppress the bioavailable amount of the polypeptide of the invention. This can be due to, for example, inhibiting the expression of components at the level of transcription, transcription stability, translation or post-translational stability. Examples of such substances are antisense RNA or double stranded interfering RNA sequences that suppress the amount of mRNA biosynthesis.

Protocols for generating cDNA libraries by reverse transcription of mRNA sequences are well known in the art, and kits for doing so are commercially available (eg from Gibco BRL). As an example, cells are selected to prepare a library of complementary DNA (ie, “cDNA”). Such cells include, but are not limited to, normal cells or cells from a subject with a disease state that is a typical disease state. As an example, if the subject has melanoma, the cell is a melanoma cell. If the subject suffers from a neurological disorder, the cell is preferably a sample of diseased cells. This approach is chosen because diseased cells are likely the best source of cDNA. That is, such molecules are specifically associated with the disease state of interest.

The preparation of an expression library is based on the established fact that messenger RNA (mRNA) must be present if the protein is expressed by cells. These mRNA molecules are not practical because they do not survive long and are unstable. Accordingly, the selected cells are used to prepare a library of complementary DNA (ie, “cDNA”). cDNA is first prepared from messenger RNA isolated from cells by reverse transcription. Protocols for generating cDNA libraries by reverse transcription of mRNA sequences are well known in the art, and kits for doing so are commercially available (eg from Gibco BRL). After preparation of the cDNA, the cDNA is used to construct a vector library. In short, the carrier vector is processed, such as by cutting and splicing, to accept the cDNA molecule. Since the engineer is familiar with many such examples, the choice of vector may vary. One commercially available vector for creating libraries is the GATEWAY ™ vector system (Invitrogen).

In vivo selection method The selection of inhibitory RNA molecules such as ribozymes is theoretically designed as usual. However, ribozymes tested in this way often do not function well in the cellular environment. Applicants here provide an in vivo selection method, which is an alternative to the method described in Applicants' WO 00/75370.

More specifically,
(I) an optional first nucleotide sequence encoding a selectable marker;
(Ii) one or more optional second nucleotide sequences, each independently encoding a marker or modulator gene, and (iii) can bind to the nucleotide sequence and effect its cleavage directly or indirectly A vector suitable for use in vivo comprising a third nucleotide sequence operably linked to a promoter encoding a gene product is provided;
(I), (ii), and (iii) are operably linked to regulatory sequences capable of directing transcription of (i), (ii), and (iii) as continuous RNA molecules in the host.

For ease of reference, this is a so-called “library vector”, ie this vector can be used to create a library of NOIs. For the purposes of the present invention, this library vector is used to generate a library of ribozymes.

In one embodiment, (ii) is misaligned with the regulatory sequence that can direct its transcription.

In a preferred embodiment, the vector comprises a single second nucleotide sequence. In a more preferred embodiment, the vector comprises two second nucleotide sequences. In a further embodiment, the vector comprises 3-5 second nucleotide sequences.

The first nucleotide preferably encodes a selectable marker gene such as an antibiotic resistance gene. Selectable markers are used to select cells transduced with the vectors of the invention.

The third nucleotide of the selection system of the present invention can be a multiple nucleotide sequence encoding an inhibitory RNA molecule. An inhibitory RNA molecule is defined as a ribonucleic acid that can inhibit transcription and / or translation of a nucleic acid sequence by binding to the sequence. In general, an inhibitory RNA molecule can bind to a nucleotide sequence or a transcript thereof and effect its cleavage directly or indirectly. Examples include ribozymes (direct cleavage), external guide sequences (EGS), and antisense or sense RNA (causing cleavage by other factors).

Further provided are vectors suitable for use in vivo, the system comprising:
(A) a first nucleotide sequence encoding a modulator gene;
(B) a second nucleotide sequence encoding a detectable marker gene and a promoter operably linked to the nucleotide sequence that the first nucleotide sequence modulates; and (c) a nucleotide sequence of interest. ,
(A), (b), and (c) are operably linked to regulatory sequences capable of directing transcription of (a), (b), and (c) as continuous RNA molecules in the host.

For ease of reference, this is a so-called “target vector”.

The target vector sequence (c) preferably comprises a gene or fragment thereof capable of encoding a polypeptide or fragment thereof, such as Parkin or Tat. This sequence is located between (a) and (b) of the vector construct, and the reading frame is shifted from the transcription unit that drives the expression of (a).

This vector is preferably a viral vector such as a lentiviral vector.

This modulator acts as an activator or inhibitor. For example, the modulator can be selected from tetracycline repressors such as TetR and tetracycline-regulated transactivators such as tTA and rtTA, and the detectable marker gene is operably linked to a tetracycline operator. be able to.

The detectable marker can be any gene product whose expression can be detected, eg, an NFAT-dependent reporter gene such as GFP or LNGFR.

In a preferred embodiment, at least one of the one or more marker genes used in the library vector is the same as the detectable marker gene used in the target vector and / or one or more used in the library vector At least one of the plurality of modulator genes is the same as the modulator gene used for the target vector and / or the promoter operably linked to the third nucleotide sequence of the library vector is the target vector ( b) The same promoter as used in the part.

The applicants further
(I) introducing a target vector and a library vector into a host cell;
(Ii) if the detection marker gene is expressed in a host cell, select that host cell, optionally,
(Iii) providing a method of selecting from a plurality of inhibitor molecules by isolating the third nucleotide sequence and determining the nucleotide sequence;

Modulator candidates, ie, modulating moieties, can be administered to non-dividing cells or slow dividing cells in any vector capable of transducing target non-dividing cells or slow dividing cells. For example, cells can be injected. Alternatively, in the case of a polypeptide candidate substance, the cell can be transfected with a nucleic acid construct that directs the expression of the polypeptide in the cell. Preferably, the expression of the polypeptide is under the control of a regulated promoter. In one embodiment, the modulating moiety is delivered to a lentiviral vector.

Vectors As is well known in the art, a vector is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present invention, by way of example, some vectors used in recombinant DNA techniques are used to replicate vectors in which entities such as DNA segments (heterologous DNA segments, heterologous cDNA segments, etc.) contain segments of DNA. Allowing it to be transferred to a host cell. Examples of vectors used in recombinant DNA techniques include, but are not limited to, plasmids, chromosomes, artificial chromosomes, or viruses.

The term “vector” includes expression vectors and / or transformation vectors.

The term “expression vector” refers to a construct capable of in vivo or in vitro / ex vivo expression.

The term “transformation vector” means a construct that can be transferred from one species to another.

Preferably, the vector is a virus-based vector.

Preferably, the vector capable of transducing target non-dividing cells or slow dividing cells is a recombinant viral vector.

In typical vectors used in the methods of the invention, at least a portion of one or more protein coding regions essential for replication can be removed from the virus. This makes the retroviral vector a replication defect. A portion of the retroviral genome can produce a vector that can transduce a target non-dividing host cell or a slow-dividing host cell and / or contains a candidate modulating moiety that can integrate the genome into the host genome. In addition, it may be replaced by a library encoding a candidate modulating portion operably linked to a regulatory control region and a reporter portion of the vector genome.

When the vector has a genome integrated into the transduced target cell and / or host cell, the transduced cell can be screened for candidate modulating moieties. Thus, transfer of the library to the site of interest in the vector genome typically involves (i) incorporating the library encoding the candidate modulating moiety into the recombinant viral vector, and (2) virion coating the modified viral vector. And (3) transducing a site of interest such as a targeted non-dividing cell or slow dividing cell, or a targeted non-dividing cell or slow dividing cell population.

Lentiviral vector Preferably, the viral vector capable of transducing target non-dividing cells or slow dividing cells is a lentiviral vector.

Lentiviral vectors are part of a larger group of retroviral vectors, and such vectors useful for this aspect of the invention are described in detail above.

The vectors of the present invention can be delivered to the target site by viral or non-viral vectors.

Non-viral delivery systems include, but are not limited to, DNA transfection methods. In the present invention, transfection includes the process of delivering a gene to a target mammalian cell using a non-viral vector.

Typical transfection methods include electroporation, DNA biolistics, lipid mediated transfection, compact DNA mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent mediated, cationic surface amphiphiles (CFA) (Nature Biotechnology, 1996, 14: 556), and combinations thereof.

Viral delivery systems include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, retrovirus vectors, lentivirus vectors, baculovirus vectors. Other examples of vectors include ex vivo delivery systems, including DNA transfection methods such as electroporation, DNA biolistics, lipid mediated transfection, compact DNA mediated transfection, etc. It is not limited.

Delivery of one or more therapeutic genes by the vector system of the present invention can be used alone or in combination with other therapies or components of therapy.

Adenoviral vectors In other embodiments of the invention, a library of candidate modulating moieties can be introduced into an adenoviral vector.

Adenoviruses are double-stranded linear DNA viruses that do not go through RNA intermediates. There are over 50 human serotype adenoviruses that fall into six subgroups based on gene sequence homology. The natural targets of adenovirus are the airway epithelium and gastrointestinal epithelium, generally producing only mild symptoms. Serotypes 2 and 5 (95% sequence homology) are most commonly used in adenoviral vector systems and are usually associated with upper respiratory tract infections in young people.

Adenoviruses are icosahedral without an envelope. Typical adenoviruses include a 140 nm encapsidated DNA virus. The icosahedral symmetry of the virus consists of 152 capsomers, 240 hexons and 12 pentons. The core of the particle contains 36 kb linear double-stranded DNA, which is covalently linked at the 5 'end with a terminal protein (TP) that acts as a primer for DNA replication. This DNA has inverted terminal repeats (ITRs) whose length varies with serotype.

Adenovirus entry into the cell involves a series of discrete events. Viral attachment to cells occurs through the interaction between viral fibers (37 nm) and fiber receptors on the cells. This receptor was recently identified for the Ad2 / 5 serotype and was named CAR (Coxacky and Adenoreceptor, Tomko et al. (1997, Proc Natl Acad Sci 94: 3352 to 2258) via cellular αvβ3 and αvβ5 integrins. Internalization of the virus into the endosome is mediated by the viral RGD sequence of the penton-based capsid protein (Wickham et al., 1993, Cell 73: 309-319) After internalization, the endosome is a process known as endosome lysis. And destroyed by events believed to be primarily facilitated by cellular αvβ5 integrin (Wickham et al., 1994, J Cell Biol 127: 257-264). In certain cell types surfaces, including skin cells and B lymphocytes buds, there is a new evidence that bind to MHC class 1α2 domain with high affinity (Hong, etc., 1997, EMBO 16: 2294~2306).

The virus then moves to the nucleus where early region activation occurs, followed immediately by DNA replication and late region activation. Transcription, replication, and packaging of adenoviral DNA requires both host and viral functional protein machinery.

Viral gene expression can be divided into early (E) and late (L) steps. A late step is defined by the onset of viral DNA replication. Adenovirus structural proteins are generally synthesized in late steps. Following adenovirus infection, host cell mRNA and protein synthesis is inhibited in cells infected with most serotypes. The adenovirus lysis cycle with adenovirus 2 and adenovirus 5 is very efficient, resulting in about 10,000 virions per infected cell in addition to excess viral protein synthesis and DNA that is not taken up by virions. Early adenoviral transcription is a complex sequential biochemical event that is interrelated, but essentially requires the synthesis of viral RNA prior to initiation of DNA replication.

The following schematic is a diagram of the adenovirus genome showing the relative orientation and location of early and late gene transcription.

The organization of the adenovirus genome is similar in all of the adenovirus groups, and specific functions are generally located at the same location for each serotype studied. Early cytoplasmic messenger RNA is complementary to four defined discontinuous regions on the viral DNA. These regions are called E1-E4. Early transcripts are classified into intermediate early (E1a), late-early (E1b, E2a, E2b, E3, and E4), and intermediate region sequences.

The early genes are expressed about 6-8 hours after infection and are driven from the seven promoters of gene blocks E1-4.

The E1a region is involved in transcriptional transactivation of viral and cellular genes and transcriptional repression of other sequences. The E1a gene performs an important regulatory function for all other early adenovirus messenger RNAs. In normal tissue, an active E1a product is required to efficiently transcribe the E1b, E2a, E2b, E3, or E4 regions. However, this E1a function can be avoided. Cells can be engineered to provide E1a-like functions, or may naturally contain such functions. This virus can also be manipulated to circumvent the E1a function. The viral packaging signal overlaps with the E1a enhancer (194-358 nucleotides).

The E1b region affects viral and cellular metabolism, and host protein arrest. This region further includes the gene encoding the pIX protein (3525-4088 nucleotides) that is required for packaging of full-length viral DNA and is important for viral thermal stability. The E1b region is required for normal progression of viral events late in infection. The E1b product acts in the host nucleus. Mutants produced within the E1b sequence exhibit reduced late viral mRNA accumulation as well as impaired host cell trafficking normally seen late in adenovirus infection. E1b is required to alter the function of the host cell so that processing and transport are favorably shifted to the viral late gene product. These products then lead to viral packaging and virion release. E1b produces a 19 kD protein that prevents apoptosis. E1b further produces a 55 kD protein that binds to p53. For a review on adenoviruses and their replication, see WO96 / 17053.

The E2 region is essential because it encodes a 72 kDa DNA binding protein, DNA polymerase, and an 80 kDa precursor of the 55 kDa terminal protein (TP) required for protein priming to initiate DNA synthesis.

A 19 kDa protein (gp19K) is encoded within the E3 region and has been implicated in the modulation of the host immune response against the virus. Expression of this protein is upregulated in response to TNF alpha during the first phase of infection, after which the protein binds and prevents migration of MHC class I antigens to the epithelial surface, thereby causing cytotoxic T lymphocytes. Suppresses recognition of adenovirus-infected cells by spheres. The E3 region is not important in in vitro studies and can be removed by deletion of the 1.9 kb XbaI fragment.

The E4 region is associated with reduced host protein synthesis and increased viral DNA replication.

There are five families of late genes, all of which start from the major late promoter. Late gene expression involves very complex post-transcriptional regulatory mechanisms, including RNA splicing. The fiber protein is encoded within the L5 region. The adenovirus genome is 103 bp in Ad5 and is flanked by inverted terminal repeats essential for DNA replication. Virus generation is complete 30-40 hours after infection.

Adenovirus can be converted for use as a vector for gene transfer by deletion of the E1 gene, which is important for induction of the E2, E3, and E4 promoters. E1 replication-defective virus can be propagated in cell lines that provide E1 polypeptide in trans, such as human embryonic kidney cell line 293. One or more therapeutic genes can be inserted by recombination instead of the E1 gene. The expression of this gene is driven from the E1 promoter or a heterologous promoter.

Further attenuated adenoviral vectors have also been developed by deleting some or all of the E4 open reading frame (ORF). However, certain second generation vectors do not appear to provide long-term gene expression, even though the DNA is believed to be maintained. Thus, it is believed that the function of one or more E4 ORFs may increase gene expression from at least certain viral promoters retained by the virus.

Another approach to creating a more defective virus was to maintain only the terminal repeats required for virus replication and completely “gut” the virus. “Gutted” or “gutless” viruses can be grown at high titers with first generation helper viruses in the 293 cell line, but they are “extracted” from the helper virus. It was difficult to isolate the vector.

Adenoviruses offer advantages as vectors for identifying candidate modulating moieties compared to other gene therapy vector systems for the following reasons.

Adenoviruses are double-stranded DNA viruses without envelopes that can transduce a wide variety of cell types of human and non-human origin in vivo and in vitro. These cells include post-mitotic terminally differentiated cells such as respiratory airway epithelial cells, hepatocytes, myocytes, cardiomyocytes, synovial cells, primary breast epithelial cells, and neurons.

Adenoviral vectors can also transduce non-dividing cells. This is very important for diseases such as cystic fibrosis where the affected cells have a slow turnover rate in the lung epithelium. Indeed, several trials are underway using adenovirus-mediated transfer of the cystic fibrosis transporter (CFTR) into the lungs of affected adult cystic fibrosis patients.

Adenoviruses have been used as vectors for gene therapy and heterologous gene expression. Large (36 kilobase) genome can accommodate foreign insert DNA of up to 8 kb, replicating efficiently in complementing cell lines, it can produce very high titers of up to 10 ^12. Thus, adenovirus is one of the best systems for studying gene expression in primary non-replicating cells.

Replicating cells are not required for the expression of viruses or foreign genes from the adenovirus genome. Adenoviral vectors enter cells by receptor-mediated endocytosis. Once in the cell, the adenoviral vector is rarely integrated into the host chromosome. Instead, adenoviral vectors function episomally in the host nucleus as a linear genome (independent of the host genome). Thus, the use of recombinant adenovirus reduces the problems associated with random integration into the host genome.

Poxvirus Vectors In other embodiments of the present invention, a library of candidate modulating moieties is introduced into a poxvirus vector.

Poxvirus vectors can be used according to the present invention because large fragments of DNA are easily cloned into their genome, and recombinant attenuated vaccinia mutants have been described (Meyer et al., 1991, J. Gen. Virol.72: 1031-1038, Smith and Moss, 1983, Gene, 25: 21-28).

Examples of poxvirus vectors include, but are not limited to, repolipoxvirus: Upton et al. Virology 60: 920 (1986) (Shoop Fibroma virus), Capripox virus: Gershon et al. Gen. Virol. 70: 525 (1989) (Kenya sheep-1), orthopoxvirus: Weir et al. Virol 46: 530 (1983) (vaccinia), Esposito et al., Virology 135: 561 (1984) (sarpox and variola virus), Hruby et al., PNAS, 80: 3411 (1983) (vaccinia), Kilpatrick et al., Virology 143: 399. (1985) (Yabasal tumor virus), avipox virus, Binns et al. Gen. Virol 69: 1275 (1988) (Tripox), Boyle et al., Virology 156: 355 (1987) (Tripox), Schnitzlein et al. Virological Methods, 20: 341 (1988) (Tripox, Quailpox), Enmopox (Lytvin et al., J. Gen. Virol 73: 3235-3240 (1992).

Pox virus vectors are widely used as expression vehicles for genes of interest in eukaryotic cells. Poxvirus vectors are widely used because of their ease of cloning and propagation in a variety of host cells, particularly for the expression of foreign proteins and as a delivery vehicle for vaccine antigens (Moss, B. 1991, Science 252: 1662). 7).

Preferred vectors for use in accordance with the present invention are recombinant poxvirus vectors such as trypox virus (FPV), entomopox virus, NYVAC and other vaccinia viruses, canarypox virus, MVA, or other non-replicating viral vector systems such as WO95. / 30018 and the like. A poxvirus vector in which at least one immune evasion gene has been deleted has also been described (see WO00 / 29428).

In a preferred embodiment, the poxvirus vector is an enmopox virus vector.

Vaccinia virus vector Preferably, the poxvirus vector is a vaccinia virus vector.

Preferably, the vector is a vaccinia virus vector such as MVA or NYVAC. Most preferred is vaccinia strain modified virus Ankara (MVA) or a strain derived therefrom. Alternatives to vaccinia vectors include avipox vectors such as tripox or canarypox, known as ALVAC, and strains derived from them that can infect human cells and express recombinant proteins but cannot replicate.

Construction of a poxvirus vector library Typically, a nucleic acid fragment or group of nucleic acid fragments encoding a candidate modulating moiety is introduced into the poxvirus genome by homologous recombination. A nucleic acid fragment or group of nucleic acid fragments encoding a candidate modulating moiety is cloned behind a vaccinia promoter adjacent to a sequence homologous to a nonessential region of the poxvirus, and the plasmid intermediate is recombined into the viral genome by homologous recombination. . This method works efficiently for relatively small inserts that are acceptable to prokaryotic hosts.

Since this method has a low frequency of homologous recombination and decreases as the size of the insert increases, construction of labor-intensive plasmid intermediates, such as library production of nucleic acid fragments, is required when large inserts are required And if the growth of DNA is not tolerated in bacteria, it is less feasible.

In order to efficiently construct a chimeric genome in a situation where homologous recombination cannot be easily performed, another method using a direct ligation vector has been developed (Merchlinsky, M. et al., 1992, Virology 190: 522). 526, Scheiflinger, F. et al., 1992, Proc. Natl. Acad. Sci. USA 89: 9777-9981). These direct ligation protocols eliminated the need for homologous recombination to produce poxvirus chimeric genomes. In such protocols, genomic-derived DNA is digested, ligated in vitro to the inserted DNA, and transfected into cells infected with helper virus (Merchlinsky, M. et al., 1992, Virology 190: 522-526, Scheiflinger). F. et al., 1992, Proc. Natl. Acad. Sci. USA 89: 9997-9981). In one protocol, the genome is digested at a unique NotI site and a DNA insert containing elements for selecting or detecting a chimeric genome is ligated to the genome arm (Scheiflinger, F. et al., 1992, Proc. Natl.Acad.Sci.USA.89: 9977-9981). This direct ligation method has been described for the insertion of foreign DNA into the vaccinia virus genome (Pfleiderer et al., 1995, J. General Virology 76: 2957-2962). Alternatively, the vaccinia WR genome removes the NotI site of the HindIII F fragment and inserts a sequence at that locus disrupts the thymidine kinase gene, allowing use of drug selection to isolate the chimeric genome. It is modified by reintroduction into the NotI site near the kinase gene (Merchlinsky, M. et al., 1992, Virology 190: 522-526).

The direct ligation vector, vNotI / tk, allows efficient cloning and propagation of DNA inserts of at least 26 kilobase pairs long (Merclinsky, M. et al., 1992, Virology 190: 522-526). Large DNA fragments are efficiently cloned into the genome, but the protein encoded by the DNA insert is expressed only at low levels corresponding to the thymidine kinase gene, an early class gene that is relatively weakly expressed in vaccinia. In addition, DNA is inserted in both directions at the NotI site.

Improved and modified vaccinia virus vectors for efficiently constructing such DNA libraries can be prepared using a “trimolecular recombination” approach that improves the efficiency of screening.

In one embodiment of the invention, a representative library of nucleic acid fragments encoding candidate modulating moieties is constructed with vaccinia virus. Preferably, a trimolecular recombination method using a modified vaccinia virus vector and an associated transfer plasmid is used to construct a library representative for vaccinia virus. This method produces nearly 100% recombinant vaccinia virus (see WO00 / 28016).

Trimolecular recombination The three molecular recombination method described above results in viral recombination close to 100%. This is a very significant improvement over current methods of transfecting vaccinia virus infected cells with plasmid transfer vectors to produce viral recombination. The latter procedure results in virus recombination with a frequency as low as 0.1%. The high yield of viral recombination in trimolecular recombination allows efficient library construction with vaccinia virus-derived vectors. Following transfection with a mixture of 20 micrograms of NotI and ApaI digested vaccinia vector arms and equimolar concentrations of tumor cell cDNA, 6 × 10 titers of recombinant virus can be obtained. This technological advance creates a new and efficient screening and selection strategy for isolating specific genomes and clones containing candidate modulating moieties.

The trimolecular recombination method disclosed herein can be used with other viruses such as mammalian viruses including vaccinia and herpes viruses. Typically, two viral arms with no homology are produced. The only way in which the viral arms can be joined is to crosslink through homologous sequences flanking the insert in a transfer vector such as a plasmid. When the two viral arms and the transfer vector are present in the same cell, only the infectious virus produced is a recombinant DNA insert of the transfer vector.

Libraries constructed in vaccinia and other mammalian viruses by the trimolecular recombination method of the invention can be used to identify candidate modulating moieties in the screening systems of the invention.

Target cell The term “target cell” includes any cell that can be derived from a suitable organism into which the vector can be transfected or transduced. Preferably, the cell is a non-dividing cell or a slow dividing cell.

Cells useful in the methods of the invention can be from any source, such as from organ cultures or in vivo, eg, from primary cultures, from established cell lines. Cell lines useful in the present invention include cancer cell lines such as fibroblast cell lines, neuroblastoma cell lines, and cell lines of blood origin. Other examples of host cells include, but are not limited to, respiratory airway epithelial cells, hepatocytes, muscle cells, cardiomyocytes, synovial cells, primary breast epithelial cells, and macrophages and / or neurons Post-mitotic terminally differentiated non-replicating cells are included. Suitable cell lines are available from the American Type Culture Collection www. atcc. org. Can be obtained from

One advantage associated with the use of immortalized cell lines derived from immortalized or slowly dividing cells of cell lines is that they have a higher probability of reflecting the in vivo state compared to the use of general cell lines. It is. In one embodiment of the invention, the target cell is a non-dividing cell or a slow dividing cell, but the target cell is immortalized immediately after or simultaneously with transduction with NOI. Immortalization can be accomplished by the addition of telomerase or hybridoma formation by fusion with neoplastic cells, or by the addition of SV40 large T antigen by protein or gene delivery.

The invention further encompasses performing the assay in transgenic animals that can be prepared according to established techniques, for example, by pronuclear injection or by preparation of ES cell chimeras.

A host cell strain can be selected that modulates the expression of the post-translational modification candidate moiety, or modifies and processes the expressed candidate modulation moiety in the specific fashion desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for the function of the protein. Different host cells have features and specific mechanisms for the post-translational processing and modification of proteins and gene products. By selecting the appropriate cell line or host system, the correct modification of the expressed foreign protein can be ensured. To achieve this goal, eukaryotic host cells with cellular mechanisms for proper processing of the primary transcript, glycosylation and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, HT1080, and WI38 cell lines.

In a preferred embodiment, the host cell is a mammalian cell.

In a preferred embodiment, the host cell is a human cell.

The present invention further provides a method comprising culturing a transduced host cell in which the cell is transduced with a vector according to the present invention under conditions suitable for the expression and / or detection of the candidate portion of the present invention.

Biological response modifier (BRM)
The pathway of interest need not be responsive to the stimulant by itself. As an example, the screening method of the present invention can be applied to a gene of interest that is constitutively expressed. In other embodiments, BRM can be used to modulate the signaling pathway under study.

Environmental stressors include, but are not limited to, one or more of oxygen depletion, radiation, heat shock, pH change, hypothermia, or glucose deprivation.

A wide range of molecules such as immunomodulators, cytokines, growth factors, cell surface receptors, hormones, mitogens, circulating molecules, inflammatory cytokines, and virulence factors such as viruses, bacteria, parasites, or yeast are used as BRMs be able to. Examples of these biological response modifiers include, but are not limited to, ApoE, ApoSAA, BDNF, cardiotrophin-1, EGF, ENA-78, eotaxin, eotaxin-2, exodas-2, Acidic FGF, basic FGF, fibroblast growth factor-10 (Marshall, 1998, Nature Biotechnology 16: 129), FLT3 ligand (Kimura et al., 1997), fractalkine (CX3C), GDNF, G-CSF, GM-CSF , GF-β1, insulin, IFN-γ, IGF-I, IGF-II, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 IL-8 (72a.a.), IL-8 (77a.a.), IL-9, IL-10, IL-11 IL-12, IL-13, IL-15, IL-16, IL-17, IL-18 (IGIF), inhibin α, inhibin β, IP-10, keratinocyte growth factor-2 (KGF-2), KGF , Leptin, LIF, lymphotactin, Mueller inhibitor, monocyte colony inhibitor, monocyte-derived protein (Marshall, 1998, supra), M-CSF, MDC (67a.a.), MDC (69a.a.), MCP-1 (MCAF), MCP-2, MCP-3, MCP-4, MDC (67a.a.), MDC (69a.a.), MIG, MIP-1α, MIP-1β, MIP-3α, MIP -3β, MIP-4, bone marrow precursor inhibitor 1 (MPIF-1), NAP-2, neuroturin, nerve growth factor, β-NGF, N -3, NT-4, Oncostatin M, PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDF1α, SDF1β, SCF, SCGF, stem cell factor (SCF), TARC, TGF-α, TGF -Β, TGF-β2, TGF-β3, tumor necrosis factor (TNF), TNF-α, TNF-β, TNIL-1, TPO, VEGF, GCP-2, GRO / MGSA, GRO-β, and GRO-γ Is included.

Stress factors such as hypoxia, heat shock, ischemia, osmotic pressure changes also induce cellular responses via signal transduction pathways, and such factors can also be used as BRMs.

Environmental stress can also be used as a BRM, including oxygen deprivation, anoxia, radiation, heat shock, pH changes, hypothermia, and glucose deprivation.

It is envisaged that overexpression of proteins that are known components of specific signaling mechanisms may be used as BRMs in addition to environmental stimuli or modulated pathways by extracellular molecules.

Stimulation specific to disease state (BRM)
The neuroapoptotic response can be induced in vitro by stimuli that can be used to mimic such disease states. Possible stimuli include hypoxia, ischemia, hypoxia / ischemia, glutamate, kainic acid, 6-hydroxydopamine, staurosporine, nitric oxide, or wortmannin.

Ischemic ischemia can be an inadequate supply of blood to a particular organ or tissue. As a result of the reduced blood supply, oxygen supply to the organ or tissue becomes inadequate (hypoxia). Prolonged hypoxia can result in injury to affected organs or tissues.

By way of example, ischemia-like conditions or conditions resulting from ischemia include hypoxia and / or low glucose concentrations.

Hypoxia As used herein, the term “hypoxia” refers to the presence of a lower than normal amount of dioxygen in a vertebrate or its blood. Inflamed tissues and solid tumors, particularly necrotic areas, are likely to have low oxygen partial pressure (<1% oxygen) or an inadequate supply of oxygen. These tissues with a low oxygen partial pressure are called hypoxic tissues. Thus, the term “hypoxia” includes both hypoxia and sites of inflammation.

Hypoxia is a potent regulator of gene expression in a wide variety of cell types (Wang and Semmnza, 1993, Proc Natl Acad Sci USA 90: 4304) and hypoxia-inducible factor 1 that binds to homologous DNA recognition sites (HIF-1) (Wang and Semmnza, 1993, supra), acting by inducing the activity of hypoxia-inducible transcription factors such as hypoxia response elements (HRE) of various gene promoters such as GAPDH and VEGF, Up-regulate gene expression. As an example, Dachs et al. (1997, supra) described mouse phosphoproteins to control the expression of both marker and therapeutic genes by human fibrosarcoma cells that respond to hypoxia within in vitro and in vivo solid tumors. Multimeric HRE derived from the glycerate kinase 1 (PGK-1) gene (First et al., 1994, Proc Natl Acad Sci USA 91: 6496) was used (Dachs et al., 1997, supra).

Nuclear accumulation of HIF-1 is slower than other transcription factors (Kallio et al., Embo J, 1998, 17: 6573-86), and up-regulation of HIF / HRE-dependent genes may take several hours ( Semenza, G. et al., J Biol Chem, 1994, 269: 23757-63), nuclear factor κB and nuclear factor IL-6 have also been implicated in the hypoxic response of several cell types (Royds et al., Mol Pathol, 1998, 51: 55-61, Matsui et al., Cardiovasc Res, 1999, 42: 104-12, Yan et al., J Biol Chem, 1997, 272: 4287-94), bZIP (basic / leucine zipper region). Induction of some members of the transcription factor class is seen in fibroblasts during anoxia Is (Estes, etc., Exp Cell Res, 1995,220: 47~54).

Alternatively, the fact that there is also significant glucose deficiency in the ischemic region of the tumor means that low glucose levels can be used to activate heterologous gene expression, particularly in tumors. A truncated 632 base pair sequence of the grp78 gene promoter known to be specifically activated by glucose deprivation has also been shown to be able to drive high level expression of reporter genes in in vivo mouse tumors ( Gazit et al., 1995, Cancer Res. 55: 1660).

Phenotypic changes The present invention involves detecting phenotypic differences between target cells and control cells. The control cell can be the target cell prior to initiating the method of the invention, i.e., in this embodiment, there is no separate control cell, and the target cell is either before or after the target cell is introduced with the modulating moiety. Cellular phenotypic characteristics are observed.

Applicants use the term “phenotype” in the usual sense, ie an observable function and structural property of an organism determined by its genetic makeup and environmental interactions.

The present invention is not limited to any particular function or structural characteristic. The following are given as examples only.

Appropriate general and specific phenotypic screening includes fundus photography, blood pressure, behavioral analysis, fluoroscopy, dual energy x-ray absorption measurement (DEXA), CAT scan, complete blood count (CBC), urinalysis , Blood chemistry, insulin levels, glucose tolerance, fluorescence activated cell separation (FACS), magnetic cell separation technology (MACS), FRET, histopathology, expression data, use of developmental biology.

The phenotypic difference may be cell death. Cytotoxicity assays can be used to study necrosis and apoptosis. There are mainly two types of cytotoxicity assays. Since dead cells become leaky, radioactive and non-radioactive assays measure an increase in permeability of the plasma membrane. Since dead cell mitochondria are unable to metabolize pigment, a colorimetric assay measures a reduction in mitochondrial metabolic activity. Apoptosis assays include those that can measure one of the following apoptosis parameters. DNA fragmentation in populations or individual cells, changes in membrane symmetry (phosphatidylserine moves from the cytoplasmic side of the cell membrane to the outside of the cell), activation of apoptotic caspases (this protein family disables many cellular functions) Release of cytochrome C and AIF into the cytoplasm by mitochondria, which triggers a cascade of events).

Methods for studying apoptosis in cell populations have focused on two major apoptotic events in cells. 1) Apoptosis and cellular cytotoxicity are characterized by cleavage of genomic DNA into discrete fragments. This DNA fragment can be assayed as a “ladder” (having a multiple of 180 bp as a “stage” of the ladder) from the cell population, for example using the Apoptotic DNA Ladder Kit or by quantifying histone complex DNA fragments by ELISA. it can. 2) Caspase activity can be analyzed by various methods including in vitro assays. The activity of a particular caspase, such as caspase 3, can be determined in cell lysates by capturing the caspase and measuring proteolytic cleavage of the appropriate substrate and by detecting cleavage of the in vivo caspase substrate. . For example, caspase 3 is activated in the initial step. Substrate PARP (poly-ADP-ribose-polymerase) and cleavage fragments can be detected by anti-PARP antibodies.

Methods for studying apoptosis in individual cells focus on 1) DNA fragmentation. The method used to assess DNA strand breaks is based on labeling and / or staining of cellular DNA. The labeled / stained DNA is then analyzed by flow cytometry, fluorescence microscopy, or light microscopy. Two different labeling methods are generally used to identify apoptotic cell DNA. An oxygen labeling method in which cellular DNA is labeled with a modified nucleotide (eg, biotin-dUTP, DIG-dUTP, fluorescein-dUTP) using an exogenous enzyme (eg, terminal transferase, DNA polymerase). This labeling method detects extensive DNA strand breaks. Enzyme labeling techniques include so-called TUNEL (TdT-mediated X-dUTP nick end labeling) enzyme labeling assays and ISNT (In Situ Nick Translation) (in situ nick translation) techniques. 2) In addition, individual cell death can also be studied by assays that measure changes in the plasma membrane (changes in symmetry or permeability of individual cell membranes that occur as the membrane contracts and gradually turns around) ). For example, during apoptosis, phosphatidylserine migrates from the cytoplasmic side of the membrane to the extracellular side and can be detected with annexin V.

Assays that measure cytotoxicity are generally based on changes in plasma membrane permeability and consequent release of components (leakage) into the supernatant, or uptake of dyes that are normally eliminated by viable cells . Alternatively, dead cells cannot metabolize various tetrazolium salts. This makes it possible to use a colorimetric assay such as MTT, XTT, or WST-1 to measure cell survival.

Individual cell survival and proliferation can be assessed by standard microscopy. For example, cells can be processed by live staining or dye exclusion and counted directly with a hemocytometer. If cells are stained differently with a fluorescent dye that binds DNA (DNA fluorescent dye), the same cell parameters can be determined by flow cytometry. Assays that observe proliferation include assays that measure DNA synthesis, i.e., a labeled DNA precursor is added to the cell culture, and cells that are approaching division take this precursor into DNA, and expression of cell cycle-related antigens Assays to monitor are included. Molecules that regulate the cell cycle are measured by their activity (eg, CDK kinase assay) or by quantification of their amount (eg, Western blot, ELISA, or immunohistochemistry).

The phenotypic difference can be, for example, a morphological change apparent from the serological properties, size, shape, color, odor, texture, and extent of attachment to the medium of the target cell.

Agar-based assays provide convenience and high throughput, and in particular provide a qualitative assessment of the potency and effectiveness of test compounds. Thus, the phenotypic difference can be the ability of cells to grow in soft agar.

A phenotypic difference can be the ability of a cell to differentiate. The ability of cells to differentiate can be determined by cell attachment to the surface in culture.

The phenotypic difference can be resistance to a drug, eg, a drug selected from the group consisting of cisplatin, doxorubicin, taxol, camptothecin, daunorubicin, and methotrexate.

The phenotypic difference can be a difference in transcription or expression of a reporter gene or cDNA. In one preferred embodiment, the phenotypic difference can be a change in the expression level of a reporter gene bound to a gene whose regulation is desired to be altered.

Screening can be done at an appropriate step in the cell cycle.

Within the scope of the present invention, secondary secondary screening can also be used, for example yeast two-hybrid vectors.

Reporter-based constructs This principle is based on the hypothesis that gene expression is controlled by its transcriptional promoter. The level of reporter gene expression (such as EGFP) may be correlated to the endogenous level of RNA for that particular gene. Cells can be functionally selected for changes in the expression level of that particular gene. For example, reporter gene levels can be compared in ribozyme library transduced and non-transduced cells. Cells can be selected in which the selected ribozyme increases selected gene promoter-mediated gene expression and increases the level of the reporter gene.

The sequence of a particular ribozyme can be used to identify a putative target gene. Thus, ribozyme-based selection systems can be used to target and identify modulators of specific promoter-driven reporter systems. This approach allows the functional selection of ribozymes that target key cellular factors involved in a particular phenotype. Furthermore, this approach allows for the identification of genes that directly regulate not only specific promoters, but any biologically relevant upstream regulators.

Construction of reporter genes Reporter genes can be constructed according to standard techniques known in the art. In general, the reporter is a coding sequence that is heterologous to the selected regulatory sequence (see below) or a modification that allows detection of the expression of the coding sequence that is normally bound to the selected regulatory sequence. In some cases, a native coding sequence usually linked to a selected regulatory sequence can be used as a reporter gene if expression of that sequence results in a detectable event.

The reporter gene can be induced directly or indirectly by an event associated with LTP induction. That is, the expression of the immediate early gene related to LTP can be used as the second signal for inducing the expression of the reporter gene. This is facilitated by the fact that some immediate early gene products are transcription factors such as zif-268 or are otherwise involved in the regulation of gene transcription. In this case, the reporter gene is operably linked to a sequence responsive to the expression of the immediate early gene product. For example, the reporter gene can be under the control of a zif-268 responsive enhancer or can include a CRE.

In a preferred embodiment of the invention, the reporter gene can be incorporated into a vector designed for DNA replication and / or transient or permanent transformation of cells and expression of the reporter gene.

As used herein, a vector (plasmid) refers to a discrete element used to introduce heterologous DNA into a cell for its expression or replication. The selection and use of such vehicles is well within the skill of the technician. Many vectors are available, and the selection of an appropriate vector depends on the intended use of the vector, i.e. whether it is used for DNA amplification or DNA expression, and the size of the DNA inserted into the vector and the characteristics of the vector. Depends on the host cell to be converted. Each vector contains various components depending on its function (amplification of DNA or expression of DNA) and the host cell in which it is compatible. Vector components generally include, but are not limited to, one or more of an origin of replication, one or more marker genes, an enhancer element, a promoter, a transcription termination sequence, and a signal sequence.

Both expression and cloning vectors generally contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically in cloning vectors this sequence is one that allows the vector to replicate independently of the host chromosomal DNA and includes origins of replication or self-replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the two plasmid origin is suitable for yeast, and the various viral origins (eg SV40, polyoma, adenovirus) are cloning vectors in mammalian cells. Useful for. In general, origin of replication components are not required for mammalian expression vectors unless used in mammalian cells suitable for high level DNA replication, such as COS cells.

Most expression vectors are shuttle vectors, that is, they can replicate in at least one class of organisms, but can be transfected into another class of organisms for expression. For example, one vector is E. coli. The same vector is then transfected into yeast or mammalian cells, although it cannot be cloned in E. coli and then replicated independently of the host cell chromosome. Alternatively, DNA can be amplified by PCR and directly transfected into host cells without replication components.

Advantageously, expression and cloning vectors may contain a selection gene, also termed a selectable marker. This gene encodes a protein necessary for the survival and growth of transformed host cells grown in selective media. Host cells that have not been transformed with the vector containing the selection gene will not survive in this medium. Typical selection genes confer resistance to antibiotics and other toxins such as ampicillin, neomycin, methotrexate, or tetracycline, supplement auxotrophic deficiencies, or supply key nutrients that are not available from complex media Encodes the protein.

As for a selection gene marker suitable for yeast, any marker gene that facilitates selection of transformants by phenotypic expression of the marker gene can be used. Suitable markers for yeast are, for example, those that confer resistance to the antibiotic G418, hygromycin, or bleomycin, or provide prototrophic properties to auxotrophic yeast mutants, such as URA3, LEU2, LYS2, TRP1 or HIS3 gene.

Vector replication is performed in E. coli. is advantageously performed in E. coli, so that E. coli is advantageously used. E. coli genetic markers and E. coli E. coli replication origin is included. They are E. E. coli plasmids such as pBR322, Bluescript (copyright) vectors, or pUC plasmids such as pUC18 or pUC19, which can be obtained from E. coli. confers resistance to antibiotics such as E. coli replication origin and ampicillin. contains both E. coli genetic markers.

Suitable selectable markers for mammalian cells are those that can identify cells that have taken up the vector, such as dihydrofolate reductase (DHFR, methotrexate resistance), thymidine kinase, or genes that confer resistance to G418 or hygromycin It is. Mammalian cell transformants are placed under selection pressure that is adapted so that only transformants that incorporate the marker and are expressed will survive independently. In the case of DHFR, or glutamine synthase (GS) markers, the transformants are transformed under conditions (at the chromosomal integration site) where the pressure gradually increases, thereby resulting in amplification of both the selection gene and the binding DNA encoding the reporter gene. By culturing, it is possible to apply a selection pressure. Amplification allows genes that are in great demand for the production of proteins important for growth to be repeated in tandem within the chromosome of the recombinant cell, along with closely related genes that may encode the desired protein. It is a process. Increased amounts of the desired protein are usually synthesized from the amplified DNA in this way.

Expression and cloning vectors generally contain a promoter that is recognized by the host organism and is operably linked to the reporter construct. Such promoters are inducible or constitutive, but in each case are subject to regulation by the regulatory sequences defined herein. By removing the promoter from the source DNA and inserting the isolated promoter sequence into the vector, the promoter can be operably linked to the DNA encoding the reporter gene. Both native promoter sequences, usually bound to a reporter, and many heterologous promoters can be used to direct expression of the reporter gene. The term “operably linked” refers to a juxtaposition that is in a relationship that allows the described components to function in the intended manner. A control sequence “operably linked” to a coding sequence is ligated in such a way that expression of the coding sequence is obtained under conditions compatible with the control sequences.

Preferably, the promoter itself is responsive to modulation by events associated with LTP. Thus, a promoter can be a promoter whose expression is derived from a gene whose expression is modulated in relation to LTP, or is modulated by the gene product of a gene whose expression is modulated in relation to LTP. Or a promoter.

Transcription of the reporter gene by higher eukaryotes can be modulated by inserting an enhancer sequence into the vector. This allows promoters that are not normally responsive to events associated with LTP induction to be responsive. Enhancers are relatively oriented and position independent. Many enhancer sequences are known from the immediate early genes that undergo LTP-related modulation and can be selected by those skilled in the art as needed.

Eukaryotic expression vectors can also contain sequences necessary to terminate transcription and stabilize mRNA. Such sequences can generally be obtained from the 5 'and 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide fragments transcribed into polyadenylated fragments of the untranslated portion of the mRNA encoding the reporter gene.

Expression vectors include any vector that can express the reporter gene described herein. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector that results in the expression of clonal DNA when introduced into a suitable host cell. Appropriate expression vectors are well known to those skilled in the art and can be replicated in eukaryotic and / or prokaryotic cells and remain episomal or integrated into the host cell genome. Is included. For example, a DNA encoding a reporter gene can be inserted into a vector suitable for cDNA expression in mammalian cells, for example, CMV enhancer-based vectors such as pEVRF (Mattias et al. (1989) NAR 17, 6418).

Useful for the practice of the present invention are expression vectors that provide for the transient expression of DNA encoding a reporter gene in mammalian cells. Transient expression usually replicates efficiently in the host cell so that the host cell accumulates many copies of the expression vector and, when stimulated, synthesizes a higher level reporter gene in turn. Use of an expression vector capable of

The construction of the vector according to the present invention uses conventional ligation techniques. In order to produce the required plasmid, the isolated plasmid or DNA fragment is cut into the desired form, adapted and religated. If desired, analysis to confirm the correct sequence in the constructed plasmid is performed in a known manner. Techniques suitable for constructing expression vectors, preparing in vitro transcripts, introducing DNA into host cells, and performing analyzes to assess reporter gene expression and function are known to those skilled in the art. It has been. The presence, amplification, and / or expression of a gene quantifies, for example, normal Southern blotting, mRNA transcription, using a suitably labeled probe that can be based on the sequences provided herein. It can be measured directly in the sample by Northern blotting, dot blotting (DNA or RNA analysis), or in situ hybridization. Those skilled in the art will readily be able to envision how these methods can be modified if desired.

Amplification According to a further aspect of the invention, the method includes the further step of amplifying the genetic factor. Selective amplification can be used as a means of enriching the genetic factor encoding the desired gene product.

In all the structures described above, the genetic material contained in the genetic element is amplified and this process can be repeated in iterative steps. Amplification can be performed by polymerase chain reaction (Saiki et al., 1988), or Q replicase amplification (Chill, Foster, and Mahan, 1991, Chetverin and Spirin, 1995, Katanaev, Kurnasov, and Spirin, 1995), ligase chain reaction (LCR) (LCR) By the use of one of a variety of other gene amplification techniques, including Landegren et al., 1988, Barany, 1991), a self-sustaining sequence replication system (Fahy, Khow, and Gingeras, 1991), and strand displacement amplification (Walker et al., 1992). Can do.

Cell Transformation Suitable cells for performing the assay according to the present invention are preferably higher eukaryotic cells derived from multicellular organisms, advantageously mammalian cells. Preferred cell types are neurons, which can be primary cultures of cells of the neural lineage or immortalized cell lines of the neural lineage.

The cells can be transformed by any suitable technique available in the art. Several techniques, such as calcium phosphate precipitation and electroporation, are described in Sambrook et al. (1989) Molecular Biology: A Laboratory Manual, Cold Spring Harbor, which is hereby incorporated by reference. Preferred cell numbers are from about 1 to about 5 × 10 ⁵ , or from about 2 × 10 ² to about 5 × 10 ⁴ . In these methods, the predetermined amount or concentration of the molecule to be tested is typically based on the amount of sample, or from about 1.0 pM to about 20 μM, or from about 10 nM to about 500 μM.

The invention further encompasses performing assays in transgenic animals that can be prepared, for example, by pronuclear microinjection or by preparing ES cell chimeras according to established techniques.

Exposure of the cells to the test compound Typically contact is for about 1 to about 24 hours, preferably about 2 to about 12 hours. Furthermore, the contact is typically made with a plurality of predetermined amounts of the molecules to be tested. In these methods, the molecule being tested can be a purified molecule, a mixture of molecules, or a homogeneous sample.

The cells or tissues to be assayed are preferably incubated with the test compound or a mixture in isotonic saline, such as Hank's balanced saline (HBSS), or artificial cerebrospinal fluid (ACSF), for the duration of exposure. Test compounds can be added to the cells by simple addition, preferably replacement with “enhancement medium” containing the compound to be tested. This medium is then advantageously replaced with a medium that supports the growth of the cell type in question. In the case of nerve cells, a nerve growth medium such as NeuroBasal (Gibco BRL) can be used. The cells are then assayed for reporter gene expression as described below.

The compound, or mixture of compounds, is advantageously contacted with cells in a high throughput screening assay. Thus, several test systems can be contacted simultaneously with different compounds, or mixtures of compounds, or with different amounts of compounds or mixtures. The effect of compound addition is measured by following the detectable signal selected for the test system used by the present invention.

LTP is induced by chemical or other means, and the activity of the test compound in modulating LTP is monitored by the assay of the present invention. For example, LTP can be induced chemically as described below, or by electric field stimulation of cultured cells. Cells can be placed in multi-well plates and stimulated using a grid to provide an appropriate electric field and LTP induction can be measured by optical screening as described.

Generation of a detectable signal A detectable signal can be generated in any one of several ways, depending on the nature of the reporter gene used in the method of the invention. For example, the detectable signal can be a luminescent signal, eg, a fluorescent signal such as GFP. GFP is a fluorescent polypeptide that generates a fluorescent signal without the need for a substrate or cofactor. GFP expression and detection techniques are well known in the art, and kits are commercially available, eg, from Clontech. GFP expression can be assayed in intact cells without the need to lyse the cells or add additional reagents. Alternatively, the detectable signal may be a signal generated as a result of enzyme activity or recognition of a cell surface marker such as LNGFR, or after selection of antibiotics, or by activation of a suicide gene, eg GCV to cells expressing TK. It can be the signal generated by the addition.

Luciferase can also be used as the basis of an assay. Luciferase expression is known in the art and kits are commercially available from Clontech (Palo Alto, Calif.). The presence of luciferase is advantageously assessed by lysing the cells, adding luciferin substrate to the cells, and then monitoring the luminescent signal by scintillation counting.

Enzyme-based assays are performed in a manner similar to luciferase-based assays, except that detection is not necessarily luminescent. The detection technique depends on the enzyme and may therefore be optical (such as in the case of β-galactosidase).

Possible Path Regulatory Components As used herein, the term “transcriptional regulatory element” includes, but is not limited to, binding to a transcription factor in a positive (induction) or negative (repression) direction. Elements that bind the gene promoter and modify its activity are included. By way of example, a “stress responsive element” or “stress responsive regulatory element” is a regulatory element that binds to a transcription factor activated by a cell in response to environmental stress.

Other examples of possible promoter regulatory control elements include, but are not limited to, tissue specific promoters, inducible promoters, physiologically regulated promoters, or any other type of optimized promoter. These promoter elements can be naturally occurring promoter elements or synthetic promoter elements. Synthetic promoter elements can be prepared by the new synthetic promoter construction method (SPCM) outlined in Edelman et al. ((2000) supra).

The control sequences of the present invention can be modified, for example, by adding additional transcription regulatory elements to make the level of transcription directed to the control sequence more responsive to the transcription modulator.

The nucleotide sequence encoding the promoter of interest is operably linked to the nucleotide sequence encoding the reporter of interest.

Functionally coupled The term “operably coupled” means in a relationship that allows the described components to function in the intended manner. A library containing regulatory sequences “operably linked” to a reporter sequence is ligated in such a way that expression of the nucleic acid reporter sequence is obtained under conditions compatible with the control sequence.

The term promoter is well known in the art and is used in the normal sense of the art, for example as an RNA polymerase binding site. The term encompasses nucleic acid regions of size and complexity ranging from a minimal promoter to a promoter including upstream elements and enhancers.

The promoter is typically selected from promoters that are functional in mammalian cells, although promoters that are functional in other eukaryotic cells can also be used. The promoter is typically derived from a viral or eukaryotic gene promoter sequence. For example, the promoter can be a promoter from the genome of the cell in which expression occurs. With respect to eukaryotic promoters, these can be promoters that function in a ubiquitous manner (eg, α-actin, β-actin, tubulin promoters, etc.) or in a tissue-specific manner (eg, pyruvate kinase gene promoters). .

Preferably, the promoter is a modified H5 or sE / L promoter (Carroll, MW, GW Wilkinson, and K Lunstrom, 2001, Mammalian expression systems and vacuation Genetic Engineers eds. See BIOS Scientific Oxford UK).

Preferably, the promoter is an early late promoter used for protein maximal production, which allows for optimum sensitivity during the antibody identification process.

One preferred promoter-enhancer combination is the human cytomegalovirus (hCMV) major immediate early (MIE) promoter / enhancer combination.

Preferably, this promoter is designed using data from Davison and Moss (J. Mol. Biol. 1989, 210: 749-769).

The level of nucleotide sequence expression under the control of a particular promoter can be modulated by manipulating the promoter region. For example, different domains within the promoter region can have different gene regulatory activities. The role of these different regions is typically assessed using vector constructs with various variants of the promoter in which specific regions have been deleted (ie, deletion analysis).

Preferably, the promoter is a hypoxia regulated promoter.

Preferably, the hypoxia regulated promoter is a cell specific hypoxia regulated promoter.

Preferably, the hypoxia regulated promoter is derived from a combination of known DNA binding moieties that is optimal for a particular cell type.

Enhancers / promoters can be primarily active in hypoxia, or ischemia, or low glucose environments when host cells are cultured under certain conditions, such as ischemic conditions. Enhancer elements, or other elements that regulate expression, can be present in multiple copies. Enhancers / promoters can be primarily active in hypoxia, or ischemia, or low glucose environments when host cells are cultured under certain conditions, such as ischemic conditions. Enhancer elements, or other elements that regulate expression, can be present in multiple copies.

Similarly, or in addition, enhancers and / or promoters are primarily active in one or more specific host cell types, such as any one or more macrophages, endothelial cells, or combinations thereof obtain.

Preferably, the tissue specific promoter is a cardiomyocyte promoter.

Preferably, the tissue specific promoter is a macrophage promoter.

Examples of suitable tissue-restricted promoter / enhancers include, but are not limited to, those that are highly active in tumor cells, such as the promoter / enhancer of the MUC1 gene, CEA gene, or 5T4 antigen gene. Examples of temporary restriction promoters / enhancers are those that are responsive to ischemia and / or hypoxia, such as hypoxia response elements or the promoter / enhancer of the grp78 or grp94 gene. The alpha fetoprotein (AFP) promoter is also a tumor specific promoter.

Preferably, the promoter of the present invention is tissue specific.

The term “tissue specific” is not limited to activity against a single tissue type, but is a promoter that exhibits selectivity in that it may be active in one group of tissues and less active or silent in another group. means. A desirable property of the promoters of the present invention is that they have a relatively low activity in the absence of an activated hypoxia regulatory enhancer element. One means of accomplishing this is to use “silencer” elements that suppress the activity of selected promoters in the absence of hypoxia.

The level of expression of one or more nucleotide sequences of interest under the control of a particular promoter can be modulated by manipulating the promoter region. For example, different domains within the promoter region can have different gene regulatory activities. The role of these different regions is typically assessed using vector constructs with various variants of the promoter in which specific regions have been deleted (ie, deletion analysis). This approach can be used, for example, to identify the minimal region that can confer tissue specificity, or the minimal region that confers hypoxia sensitivity.

Some of the tissue specific promoters described above may be particularly advantageous for the practice of the present invention. In most cases, these promoters can be isolated as convenient restriction digest fragments suitable for cloning in the selected vector. Alternatively, promoter fragments can be isolated using the polymerase chain reaction. Cloning of the amplified fragment can be facilitated by incorporating a restriction site at the 5 'end of the primer.

Preferably, the ischemia responsive promoter is a tissue restricted ischemic responsive promoter.

Preferably, the tissue restricted ischemia responsive promoter is a macrophage specific promoter restricted by repression.

Preferably, the tissue restricted ischemia responsive promoter is an endothelium specific promoter.

Preferably, the tissue restricted ischemia responsive promoter of the present invention is an ILRE responsive promoter.

For example, glucose-regulated proteins (grp) such as grp78 and grp94 are highly conserved proteins known to be induced by glucose deprivation (Attenello and Lee, 1984, Science 226, 187-190). The grp78 gene is expressed at low levels in most normal healthy tissues under the influence of basal levels of promoter elements, but has at least two important “stress-inducible regulatory elements” upstream of the TATA element (Attenello, 1984, supra, Gazit et al., 1995, Cancer Res 55: 1660-1663). Addition of the grp78 promoter to the 5'-end truncated 632 base pair sequence confers high inducibility to glucose deficiency on the reporter gene in vitro (Gazit et al., 1995, supra). Furthermore, this promoter sequence in retroviral vectors was able to drive high levels of expression of the reporter gene in mouse fibrosarcoma, particularly tumor cells in the central relatively ischemic / fibrotic site (Gazit et al., 1995, supra).

Preferably, the selected physiologically regulated promoter element is a heat shock promoter element.

Preferably, the selected physiologically regulated promoter element is a radiation element.

Preferably, the selected physiologically regulated promoter element is an ischemic element.

Enhancers Furthermore, these promoters of the invention can be modified by adding additional regulatory sequences, such as enhancer sequences. Chimeric promoters comprising sequence elements from two or more different promoters as described above can also be used.

The term “enhancer” includes DNA sequences that bind to other protein components of the transcription initiation complex, thereby facilitating the initiation of transcription directed by a bound promoter.

Other examples of suitable tissue-restricted promoter / enhancers are those that are highly active in tumor cells, such as the MUC1 gene, CEA gene, or 5T4 antigen gene promoter / enhancer. The alpha fetoprotein (AFP) promoter is also a tumor specific promoter. One preferred promoter-enhancer combination is the human cytomegalovirus (hCMV) major immediate early (MIE) promoter / enhancer combination.

A preferred enhancer is the hypoxia response element (HRE).

HRE
As mentioned above, hypoxia is a potent regulator of gene expression in a wide variety of cell types, and hypoxia-inducible factor 1 (HIF-1, Wang and Semenza, 1993, which binds to homologous DNA recognition sites. Proc Natl Acad Sci, 90: 430), acting by inducing the activity of hypoxia-inducible transcription factors such as hypoxia response elements (HRE) of various gene promoters. Dachs et al. (1997, Nature Med 5: 515) have described mouse phosphoproteins to control the expression of both marker and therapeutic genes by human fibrosarcoma cells that respond to hypoxia within in vitro and in vivo solid tumors. A multimeric HRE derived from the glycerate kinase 1 (PGK-1) gene (First et al., 1994, Proc Natl Acad Sci, 91: 6496-6500) was used (Dachs et al., Supra).

Hypoxia response enhancer element (HREE) has also been found in association with several genes including the erythropoietin (EPO) gene (Madan et al., 1993, Proc Natl Acad Sci 90: 3928, Semenza and Wang, 1992, Mol Cell Biol, 1992, 12: 5447-5454). Other HREEs are the muscle glycolytic enzyme pyruvate kinase (PKM) gene (Takenaka et al., 1989, J. Biol Chem 264: 2363-2367), the human muscle specific β enolase gene (ENO3, Peshavaria and Day, 1991, Biochem). J 275: 427-433) and the regulatory region of the endothelin-1 (ET-1) gene (Inoue et al., 1989, J Biol Chem 264: 14954-14959).

Preferably, the HRE of the present invention includes, for example, erythropoietin HRE element (HREE1), muscle pyruvate kinase (PKM), HRE element, phosphoglycerate kinase (PGK) HRE, β-enolase (enolase 3, ENO3) HRE element , Endothelin 1 (ET-1) HRE element, and metallothionein II (MTII) HRE element.

Agents The expression and / or detection of candidate target portions and / or modulating portions of the present invention can be carried out using agents capable of directly or indirectly recognizing the candidate portions. Agents can include, but are not limited to, samples obtained from a subject or animal, or samples that can include tissue samples or body fluid samples. Not.

As used herein, the term “tissue” refers to any biological object composed of a single cell, multiple cells, a cluster of cells, or an entire organ. The term tissue further encompasses one or more cells that can be normal or abnormal (ie, a tumor). A “body fluid” can be any liquid substance extracted, excreted or secreted from an organism or tissue of an organism. Body fluids do not necessarily contain cells. Body fluids relevant to the present invention include, but are not limited to, whole blood, serum, plasma, urine, cerebrospinal fluid, tears, and amniotic fluid.

Binding partner (BP)
Detection of the expression of candidate targets and / or modulating moieties of the present invention can be achieved, for example, by the application of a binding partner that can specifically react with the reporter moiety of the present invention.

Preferably, the binding partner (BP) comprises one or more binding domains capable of detecting the reporter moiety and / or binding to one or more host cells expressing the reporter moiety. Thus, BP is directed to specific cells by affinity for the reporter moiety.

One or more binding domains of BP consist, for example, of a natural ligand of a reporter moiety, which retains binding affinity for an adhesion molecule, or a growth factor receptor ligand (eg, epidermal growth factor), or a reporter moiety. Fragments of natural ligands.

Alternatively, the binding domain can be derived from the heavy and light chain sequences of an immunoglobulin (Ig) variable region. Such a variable region can be derived from a natural human antibody or another species of antibody such as a rodent antibody. Alternatively, the variable regions can be derived from engineered antibodies such as humanized antibodies, or phage display libraries of immunized or non-immunized animals, mutant phage display libraries. As another alternative, the variable region can be derived from a single chain variable fragment (scFV). BP can contain other sequences to achieve multimerization or to act as a spacer between binding domains, or due to restriction site insertions in the gene encoding BP, and Ig hinge sequences Or a new spacer and an engineered linker sequence.

A BP can include all or part of an Ig heavy chain constant region in addition to one or more immunoglobulin variable regions, so that natural whole Ig, engineered Ig, engineered Ig-like molecule, Single chain Ig or single chain Ig-like molecules can be included. Alternatively, or in addition, BP can contain one or more domains from other proteins such as toxins.

Antibody Preferably the binding partner is an antibody.

As used herein, “antibody” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Antibodies can exist as several fragments, including intact immunoglobulins or well-characterized fragments produced by digestion with various peptidases. Although various antibody fragments have been defined for digestion of intact antibodies, one skilled in the art will understand that antibody fragments can be synthesized from scratch either chemically or using recombinant DNA methods. Thus, as used herein, the term antibody further includes antibody fragments produced by modification of whole antibodies or synthesized from the beginning using recombinant DNA methods. Antibody fragments encompassed by use of the term “antibody” include, but are not limited to, Fab, Fab ′, F (ab ′) 2, scFv, Fv, dsFv bispecific antibodies, and Fd Fragments are included.

Antibodies that are specifically immunoreactive with the reporter moiety of the present invention are another embodiment of the present invention. These antibodies can be monoclonal or polyclonal. These antibodies may optionally be recombinant or purely synthetic. The antibody can be an intact antibody or a fragment. Preparation of antibodies specific for the candidate portion of the invention is a routine procedure for those skilled in the art.

In the present invention, an antibody array can be used. The antibodies on the array can be monoclonal or polyclonal. The antibodies on the array can be intact antibodies, or fragments of antibodies, that can specifically bind to a reporter moiety of the invention. Preferably, the antibody array comprises at least 4 different antibodies, preferably more than about 10 different antibodies. For example, a method for detecting and / or assaying reporter moiety expression involves first contacting a sample of body fluid or tissue obtained from an animal or an antibody array with a clone from a library of candidate modulating moieties of the invention. Includes steps. The reporter moiety can be contacted with an animal tissue or fluid sample, or directly with the antibody array, and binding between the reporter moiety and the antibody on the array can be detected. Alternatively, tissue or body fluid samples can be purified and antibody or mRNA transcripts isolated prior to contacting the clone.

Preferably, the antibody is a polyclonal antibody.

Preferably, the antibody is a monoclonal antibody.

Preferably, the antibody is high affinity and high titer.

If the antibody is of low affinity and / or titer, preferably the reporter moiety is expressed at a high level.

Screening of candidate candidate modulating moieties Direct or indirect detection of the expression of the reporter moiety of the present invention can be achieved, for example, by applying a labeled antibody that is specifically immunoreactive with the expression product of the reporter moiety. This antibody can be obtained from a tissue or body fluid sample removed from a human or animal. Various forms of typical immunoassays known to those skilled in the art can be applied thereto. These assays include both competitive and non-competitive assays. For example, in one assay format, sometimes referred to as a “sandwich assay,” an immobilized antibody that specifically reacts with a reporter moiety is contacted with a biological tissue or fluid sample. The presence of the immobilized reporter moiety antibody complex can then be achieved by applying an immunoreactive second labeled antibody to the reporter moiety or reporter moiety-antibody complex. In other embodiments of the invention, Western blot format assays may also be used.

Use of identified target and / or modulating moiety The identified target and / or modulating moiety of the present invention is operably linked to a regulatory control region and / or a nucleotide sequence of interest (NOI). It can be used in vectors. In one embodiment, the identified target and / or modulating moiety of the present invention is operative in a regulatory control region and / or nucleotide sequence of the vector to deliver the vector to, for example, a target cell or target tissue. Can be linked.

In one aspect of the pharmaceutical composition, the present invention comprises a vector comprising an identified modulating moiety operably linked to a regulatory control region and / or NOI, and a pharmaceutically acceptable carrier, diluent, or enhancer. Pharmaceutical compositions comprising the forms (including combinations thereof) are provided.

The pharmaceutical composition can be used for humans or animals in human and veterinary medicine and typically comprises one or more pharmaceutically acceptable diluents, carriers or excipients. Carriers or diluents acceptable for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Ltd. (A. R. Gennaro, 1985). The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition can include any suitable binder, lubricant, suspending agent, coating, solubilizer, as or in addition to a carrier, excipient or diluent.

Preservatives, stabilizers, dyes, and flavoring agents can also be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

There may be different composition / formulation requirements depending on the different delivery systems. By way of example, the pharmaceutical composition of the present invention can be obtained using a minipump or by a mucosal route, for example as a nasal spray or aerosol for inhalable or ingestible solutions, or by a composition such as intravenous, intramuscular or subcutaneous route. It can be formulated to be delivered parenterally that is formulated in an injectable form for delivery. Alternatively, the formulation can be designed to be delivered by both routes.

If the pharmaceutical composition is delivered by mucosal route via the gastrointestinal mucosa, it should be able to maintain stability during transport through the gastrointestinal tract, e.g. resistant to proteolysis and stable at acidic pH Should be resistant to the cleaning action of bile.

Where appropriate, the pharmaceutical compositions may be obtained by inhalation, in the form of suppositories or vaginal suppositories, topically in the form of lotions, solutions, emulsions, ointments or powders, by the use of skin patches, starch or lactose. Or in the form of tablets containing excipients such as chalk, or in the form of capsules or eggs, alone or mixed with excipients, or elixirs, solutions, or suspensions containing fragrances or colorants Or the pharmaceutical composition can be injected parenterally, for example, intravenously, intramuscularly, or subcutaneously. For parenteral administration, the composition is best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood. obtain. For buccal or sublingual administration, the compositions can be administered in the form of tablets or lozenges that can be formulated in conventional manner.

Administration Typically, a physician will determine the appropriate actual dosage for an individual subject, which will depend on the age, weight, and response of the particular patient and the severity of the disease. The following dosage is an example of an average case. Of course, there may be individual cases where higher or lower dosages are beneficial.

The composition (or component thereof) of the present invention can be administered orally. Additionally or alternatively, the compositions (or components thereof) of the present invention can be administered by direct injection. Additionally or alternatively, the compositions (or components thereof) of the present invention can be administered topically. Additionally or alternatively, the compositions (or components thereof) of the present invention can be administered by inhalation. Additionally or alternatively, the composition (or component thereof) of the present invention is further administered by one or more of parenteral, mucosal, intramuscular, intravenous, subcutaneous, intraocular, or transdermal means of administration. And can be formulated for such administration.

As a further example, the pharmaceutical composition of the present invention can be administered according to a dosing schedule such as from 1 to 10 times daily, for example once or twice daily. The particular dose level and dosing frequency for any particular patient can vary, including the activity of the particular compound used, the metabolic stability and duration of action of that compound, age, weight, general health It depends on a variety of factors, including sex, diet, mode of administration and time, excretion rate, drug combination, severity of the particular disease, and the treatment the host is receiving.

The term “administration” further includes, but is not limited to, delivery by mucosal route, eg, as a nasal spray or aerosol for inhalation, or as an ingestible solution, eg, intravenous, intramuscular, or subcutaneous route, etc. In the case of delivery by injectable form, parenteral delivery is included.

Accordingly, the pharmaceutical composition of the present invention is administered orally, injection (direct injection, etc.), topical, inhalation, parenteral administration, mucosal administration, intramuscular administration, intravenous administration, subcutaneous administration, intraocular administration, or transdermal administration. Can be administered by one or more routes.

Pharmaceutical compositions comprising an effective amount of a vector comprising an identified modulating moiety operably linked to a disease NOI are used for the treatment of diseases such as those listed in WO-A-98 / 09985 be able to. For ease of reference, a portion of the list is shown below. Macrophage inhibitory and / or T cell inhibitory activity and thereby anti-inflammatory activity; anti-immune activity, ie inhibitory action on cells and / or humoral immune responses including responses not related to inflammation; viruses and / or other intracellular pathogens Diseases related to macrophages; ability to adhere to extracellular matrix components and fibronectin of macrophages and T cells, and inhibition of upregulated fas receptor expression in T cells; inhibition of unwanted immune responses and inflammation, including rheumatoid arthritis Arthritis, inflammation associated with hypersensitivity, allergic reaction, asthma, systemic lupus erythematosus, antigenic disease and other autoimmune diseases, inflammation associated with atherosclerosis, arteriosclerosis, arteriosclerotic heart disease, reperfusion injury, cardiac arrest For myocardial infarction, vascular inflammatory disease, respiratory distress syndrome or other cardiopulmonary disease, peptic ulcer Urinary inflammation, ulcerative colitis, and other gastrointestinal diseases, liver fibrosis, cirrhosis or other liver diseases, thyroiditis or other glandular diseases, glomerulonephritis or other kidney and urological diseases, otitis or other ENT diseases, dermatitis or other skin diseases, periodontal disease or other dental diseases, testicularitis or testicular-epididymis, infertility, testicular trauma or other immune-related testicular diseases, placental dysfunction, placental function Failure, habitual miscarriage, eclampsia, preeclampsia and other immune and / or inflammation-related gynecological diseases, posterior uveitis, middle uveitis, anterior uveitis, conjunctivitis, chorioretinitis, uveal retina Inflammation, optic neuritis, intraocular inflammation, such as retinitis or cystoid macular edema, sympathetic ophthalmitis, scleritis, retinitis pigmentosa, immune and inflammatory components of degenerative fundus disease, inflammatory components of ocular trauma, eyes due to infection Inflammation, proliferative vitreoretinopathy, acute ischemic optic neuropathy, For example, excessive scarring after glaucoma filtration surgery, immune and / or inflammatory response to ocular grafts, and other immune and inflammation related eye diseases, suppression of immunity and / or inflammation in the central nervous system (CNS) or other organs Inflammation associated with beneficial autoimmune diseases or conditions or disorders, Parkinson's disease, complications and / or side effects of Parkinson's disease treatment, AIDS-related dementia complex HIV-related encephalopathy, Devic's disease, Sydenham chorea, Alzheimer's disease and other CNS Degenerative disease, condition or disorder, inflammatory component of Stokes, post-polio syndrome, immune and inflammatory component of mental disorder, myelitis, encephalitis, subacute sclerosing panencephalitis, encephalomyelitis, acute neuropathy, subacute neuropathy, Chronic neuropathy, Guillain-Barre syndrome, Sydenham chorea, myasthenia gravis, pseudobrain tumor, Down syndrome, Huntington's disease, atrophic lateral cord Sclerosis, inflammatory component of CNS compression or CNS trauma or CNS infection, inflammatory component of muscle atrophy and dystrophy, immune and inflammation related diseases, conditions and disorders of the central and peripheral nervous system, post-traumatic inflammation, septic shock, Associated with infection, inflammatory complications or side effects of surgery, bone marrow transplantation or other transplant complications and / or side effects, eg inflammation and / or immune complications and side effects of gene therapy due to infection from a viral carrier, or AIDS Suppression or inhibition of inflammation, humoral and / or cellular immune response, treatment or alleviation of monocyte or leukocyte proliferative diseases such as leukemia, by reducing certain amounts of monocytes or lymphocytes, natural or artificial cells, Prevention of graft rejection during transplantation of tissues and organs, such as cornea, bone marrow, organ, lens, pacemaker, natural or artificial skin tissue Beauty / or a treatment. Specific cancer-related diseases include, but are not limited to, solid tumors; hematologic tumors such as leukemia; tumor metastases; benign tumors such as hemangiomas, acoustic neuromas, neurofibromas, trachoma, and suppuration Rheumatoid arthritis; rheumatoid arthritis; psoriasis; ocular angiogenic diseases such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal transplant rejection, neovascular glaucoma, post-lens fiber proliferation, lebeosis; Osler-Weber syndrome; Angiogenesis; plaque-forming neovascularization; telangiectasia; hemophilia arthropathy; angiofibroma; wound granulation; coronary accessory; cerebral accessory; arteriovenous malformation; ischemic limb neovascularization; Neonatal glaucoma; Post lens fiber growth; Diabetic neovascularization;

The invention will now be described, by way of example only, with reference to the following drawings.

Identification of steps and target genes in the identification of cell response and modulation parts (using expression profiling)
• Identification of target cells • Introduction of a construct containing a promoter (and enhancer) operably linked to a reporter gene (eg, GFP, LNGFR) into the target cell • Cell culture and appropriately transduced cells • Cell transduction with the library of interest • Cell screening under conditions that activate the cell response pathway and produce a detectable phenotype (eg, increased reporter expression / transcription, cell death, etc.) Collection / isolation of cells that show phenotypic changes, library insertion, amplification ・ This enables identification of cell response and modulation parts ・ Optional modulation part retest

FIG. 1 is a schematic diagram of the method of the present invention.

Step 1 Identification of Target NOIs The present invention provides screening methods for isolating unidentified components of signal transduction pathways that are adapted to the cellular response pathway of interest.

In this example, this approach requires prior identification of a NOI whose expression is specifically up-regulated or down-regulated in response to a stimulus that activates the pathway (or its withdrawal).

If the appropriate target NOI is not known, the target NOI can be a candidate identified by expression profiling.

Step 2 Preparation of the construct A construct comprising a NOI promoter operably linked to a reporter gene is prepared. As an example, the NOI promoter is then used to drive the expression of a reporter gene such as GFP or LNGFR.

Step 3 Identification of target cells of interest Identify target cells and transfect this construct with a construct consisting of a reporter cDNA flanked by sufficient sequences to allow homologous recombination at the site of interest. Can be introduced into target cells (Hanson and Sedivy, 1995, Mol cell Biol 15 (1): 45-51). This approach has the advantage that a full-length promoter including any enhancer drives reporter expression.

Alternatively, the expression cassette can be cloned into a viral vector and used to transduce the target cells of interest (providing target cells that are stably expressed).

Step 4 Identification of appropriately transfected or transduced target cells Transfected / transduced cells are maintained under appropriate cell culture conditions that allow identification of transfected / transduced cells.

Step 5 Introduction of the library of interest into the transduced cells Once the stably transfected / transduced cells are cloned or cells expressing similar GFP levels are pooled, the ribozyme library or antisense A library of modulating moieties, such as a library, can be introduced into target cells by transfection or transduction. A schematic diagram of the library vector is shown in FIG.

This vector genome can be designed for the purpose of studying specific conditions.

Constitutive expression of the encoded cDNA This vector genome is constitutive of expression of the encoded cDNA (this technique is described in Applicants' WO 98/17816, WO 99/32646, and WO 98/17815). Or limited to only transduced cells (this technique is as described in Applicants et al. WO 99/15683).

Limiting gene expression to only transduced cells allows construction of the library in bacterial hosts and subsequent production of lentiviral vectors in eukaryotic cells, while simultaneously providing a given cell or Samples representing all genes expressed in the tissue are maintained. This avoids the problems associated with obtaining a library representing a toxin gene since unregulated expression does not occur during construction of the library or during production of the lentiviral vector.

This vector genome can incorporate the GATEWAY ™ cloning technology (Gibco BRL) to enable rapid cloning of the commercially available GATEWAY ™ library of interest into the vector.

Briefly, this library can be constructed into GATEWAY ™ entry clones selected in view of the required outcome. The lentiviral genome can be adapted to GATEWAY ™ technology by incorporating a conversion cassette as described by the manufacturer.

The use of GATEWAY ™ technology facilitates subsequent identification of the gene of interest, for example, the use of other GATEWAY ™ compatible vectors can be used to determine the mechanism of action of a given gene. A rapid means of subsequent screening and confirmation can be provided (eg, yeast two-hybrid vectors).

This vector genome can incorporate a promoter cassette that facilitates rapid modification of the promoter and / or enhancer region involved in gene expression. Thereby, the promoter in the vector genome is selected considering the target cell / tissue type. For example, to study the effects of library genes on tissues during hypoxic response, a hypoxic response element (HRE) is incorporated into the enhancer region of the vector promoter and expression of the gene of interest under such experimental conditions Can be strengthened.

Once the cell / tissue type / library combination has been selected, the library of interest can be cloned into a GATEWAY ™ compatible lentiviral genome containing the necessary promoter / enhancer cassette, which is the GATEWAY ( Performed using the LR reaction as outlined in the user manual of the Cloning Technology (Gibco BRL).

Another important consideration for designing an appropriate vector genome is to ensure that the gene of interest from transduced cells can be recovered quickly. For this, the vector specific region of DNA is designed to flank the integrated gene / promoter cassette, so that it can be extracted from cellular DNA extracts by PCR using oligonucleotide primers specific for that flank region. The gene can be recovered.

Further confirmation of the gene of interest is facilitated by simple and rapid transfer of such genes to other GATEWAY ™ vectors. This is done by the alternating reaction of LR and BP as described in the GATEWAY ™ manual.

After production of the lentiviral library virus, it is used to transduce cells. These can be cells that have been exposed to specific environmental conditions or tissues that are in a specific disease state. Transduced cell / tissue analysis aims to detect altered cellular responses to responses that normally occur in a given environmental or disease state.

Similar approaches to those described above for lentiviral vector libraries can be designed for other vector systems. Examples of other possible vector systems are retrovirus (eg MoMLV) based systems, adenovirus based systems, or adeno-associated virus-2 (AAV-2) based systems.

Step 6 Screening Transfect / Transduced Cells The transfected / transduced cells are then screened under conditions that activate the cellular response pathway and thereby activate reporter expression. As an example, in the case of promoters that are down-regulated by pathway activation, transduced cells are screened for GFP switch-on.

Step 7 Isolation of cells expressing the phenotype of interest Identified cells expressing the phenotype of interest (eg, up-regulation or down-regulation of GFP expression) are collected by FACS and the library insert is Amplify by PCR using region primers. Candidates are further tested by retransduction of target cells.

Inducing / maintaining cell division of target cells that may be immortalized terminally differentiated or primary cells of limited proliferative potential (which may also be described as non-dividing or slow division / senescence / pause) Transduction with a lentiviral vector encoding NOI that can be done (immortalized). NOI can be one or more “immortalized genes”, for example, ectopic expression of a combination of human telomerase (hTERT) and a temperature-sensitive mutant of a simian virus large tumor antigen has been newly isolated. It has been shown that conditionally immortalized breast fibroblasts and endothelial cells (O'Hare, MJ, et al., Proc Natl Acad Sci USA (January 16, 2001) 98 (2) : 646-51). If the inhibitory protein prevents cell division, the NOI can include a ribozyme.

The aim is to reproduce the in vivo cellular environment as closely as possible while facilitating the screening of engineered cells.

The in vitro cultured cells are listed below in the order of "desirability" in descending order of similarity to in vivo cells, and in ascending order of ease of handling. Is the balance.
1. 1. In vitro primary cells 2. Primary cells that have been “immortalized” by delivery of the appropriate NOI by a lentiviral vector. Established immortal cell line

Although current technology limits target cells to dividing cells, the present invention allows screening of all these cell types.

Identification of drug targets and modulating moieties involved in cancer response pathways such as breast cancer Neu-Ras pathway Breast cancer is the most common malignancy in women. Cyclin D1 is overexpressed in most breast cancers (Gillett et al., 1994, Cancer Res 54 (7): 1812-7). In breast epithelial cells, the Neu-Ras pathway is linked to the cell cycle apparatus by cyclin D1 (Yu et al., 2001, Nature 411 (6841): 1017-21), which is absolute to the malignant transformation of this tissue It is a requirement. Anti-cyclin D1 therapy is believed to have great potential in the treatment of human breast cancer with Neu-Ras pathway activation. The methods of the invention can be used to identify targets other than cyclin D1 that carry Neu-Ras signals that result in inappropriate up-regulation of the cyclin D1 promoter.

Preparation of a construct for introduction into target cells A construct comprising a cyclin D1 promoter operably linked to a reporter gene is prepared.

Since mammary epithelial cells do not grow in the absence of cyclin D1, cells other than mammary epithelial cells can be used in the selection procedure. Potential target cells include, but are not limited to, Ras and Neu-driven fibroblast tumors because they express high levels of cyclin D2 and cyclin D3 in addition to cyclin D1. The removal of cyclin D1 does not interfere with cell growth. The selection of these cells has the advantage of directing the selection towards a cyclin D1-specific target while the expression of cyclin D2 and / or cyclin D3 remains unaffected. Also suitable are cells such as HT1080, an immortalized human fibroblast cell line.

Activation of the cellular maintenance pathway under conditions that activate the response pathway can be achieved by overexpression of Ras or Neu (upstream of Ras). Reporter expression may depend on the stage of the cell cycle. Therefore, this screening method can be adapted so that screening is usually performed at a stage (eg, G1) in the cell cycle where cyclin D1 is highly expressed. Alternatively, target cells can be synchronized prior to screening by block and release, or elutriation. Another option is to perform continuous screening so that the probabilities of all cells being screened at the appropriate time in the cell cycle match.

Identification of drug targets and modulating moieties involved in inflammatory disease response pathways such as asthma. A summary of lentiviral methods for identifying drug targets for inflammatory diseases is shown in FIG.
Inflammatory Disease Many inflammatory disease states are thought to be due to an imbalance in the T helper cell population (Stirling and Chung, 2000, Eur Respir J 16 (6): 1158-74). The identification of targets in signal transduction pathways that lead to Th2 differentiation but not Th1 differentiation of CD4 progenitor cells proves to be beneficial in a range of inflammatory disease types.

Asthma The asthmatic response is a multifactorial response pathway characterized by upregulation of eosinophil levels. IL-5 receptors are expressed in cells of the eosinophil lineage and act to induce eosinophil differentiation and prolong their survival. The asthmatic response is also thought to be due to an imbalance in the T helper cell population. Therefore, intervention in one stage below may help reduce eosinophilia.

Possible interventions include, but are not limited to,
Inhibition / blocking of the IL-5 pathway in eosinophils. Reduction of circulating Th2 cells by expression of Th2-derived cytokines, eg inhibition of IL-5 inhibition Th2 differentiation is included.

Glucocorticoids also act to repress IL-5 promoter transcription in Th2 cells, but are known to have undesirable side effects (Weltman and Karim, 2000, Expert Opin Investig Drugs 9 (3): 491- 6). Thus, the identification of proteins that result in IL-5 transcription of T cells may also provide another drug target that prevents IL-5 secretion by T cells.

IL-5 Signaling Pathway IL-5 receptors are expressed in cells of the eosinophil lineage and act to induce eosinophil differentiation and prolong their survival. Eosinophilia is a feature of asthma and inhibition of the IL-5 signaling pathway should alleviate the symptoms of the disease.

Preparation of constructs using IL-5 promoter The promoter of NOI up-regulated by IL-5 is used to drive expression of the reporter gene in the appropriate cell type.

Identification of suitable target cells Appropriate IL-5 dependent target cells are selected, such as the TF-1.8 cell line. Alternatively, the use of target T cells such as Th1 and Th2 cells may be able to identify targets in signaling pathways that lead to Th2 differentiation rather than Th1 differentiation of CD4 progenitor cells, which is beneficial in a range of inflammatory disease types .

Identification of drug targets and modulating moieties involved in neurodegenerative disease response pathways such as Alzheimer's disease Alzheimer's disease is characterized by Aβ-amyloid deposition and neurofibrillary tangles in many brain regions. Two β-secretases, BACE1 and BACE2, are involved in the production of toxic Alzheimer's disease Aβ peptides. They are produced by endoprotease cleavage of β-amyloid precursor protein (APP) by β and γ-secretase. Secretion of Aβ peptide has been reported to be eradicated in cultures of BACE-1 deficient cortical neurons (Cai, H. et al., Nat Neurosci (March 2001) 4 (3): 233-4). In the brain, BACE1 mRNA is at a high level. BACE1 is synthesized as a proenzyme, which is cleaved by furin to produce the mature enzyme. There is no apparent adverse effect associated with BACE1 deficiency in mice, and thus it has been shown that inhibition of BACE1 in humans may not have mechanism-based toxicity (Luo et al., 2001).

Preparation of the construct The BACE1 promoter is selected to drive reporter expression.

Target cells Target cells are neurons.

Maintenance of target cells In this case, reporter expression is constitutive.

Transduction of the library of interest Following transduction with the library (antisense, cDNA, or ribozyme), cells are screened for reporter switch-off.

Identification of cells expressing the phenotype of interest A molecule that eliminates reporter expression, and thus BACE1, is a potential therapeutic / drug target for Alzheimer's therapy.

A similar method can be followed for γ-secretase.

Target genes Genes that interfere with the apoptotic response

Disease state Hypoxia-induced apoptosis in Parkinson's disease neurons

Target / host cell Rat neuron

Principle Neuronal apoptosis is an important factor in the pathogenesis of several neurological diseases including Parkinson's disease.

Stimulated neuronal apoptotic responses in vitro can be induced in vitro by stimuli that can be used to mimic such disease states. Possible stimuli include hypoxia, ischemia, hypoxia / ischemia, glutamate, kainic acid, 6-hydroxydopamine, staurosporine, nitrogen monoxide, or wortmannin.

Apoptosis can be rapidly assessed by histology and immunohistochemistry combined with molecular assays such as caspase-3 activation, DNA laddering, or cell membrane permeabilization, and cells that survive hypoxia-induced apoptosis are non-apoptotic cells. Show.

For example, to identify genes with receptive ability to prevent hypoxia-induced apoptosis in neurons, a cDNA library is constructed into a lentiviral vector (eg, a representative rat nerve (or other rat cell type / tissue) cDNA Library).

Vector design genes are expressed under the control of a minimal CMV promoter and HRE.

Target host cells Rat neurons are transduced with this lentiviral vector library.

Identification of the gene of interest Cells that survive in hypoxia-induced apoptosis are analyzed in a PCR-based assay designed to amplify the cDNA expressed by this transduction vector.

Once the secondary screening gene using the vector genome plus gene has been isolated, sequenced, and identified, the ability of the gene to reconstruct the vector genome and inhibit both hypoxia-induced apoptosis and apoptosis induced by other stimuli Design secondary screens to test

Further studies are conducted to characterize the mode of action of the gene under study and to identify possible uses of the gene in therapy or other fields.

Use of lentiviral vectors to identify genes that allow human eosinophils to survive in vitro in the absence of IL-5. Eosinophils, and eosinophil cell lines such as TF-1, are in vitro. Rely on exogenous IL-5 for survival in Removal of IL-5 from these cells results in rapid cell death.

cDNA library A library that expresses cDNA isolated from the eosinophil cell line AML14.3D10. This cell line is independent of exogenous IL-5 for its survival.

Vector design lentiviral cDNA library constructs a vector (eg a library expressing cDNA isolated from the eosinophil cell line AML14.3D10. This cell line is independent of exogenous IL-5 for its survival )

Target host cell IL-5 dependent eosinophils or TF-1 cells are transduced with this library to remove exogenous IL-5.

Screening Cells that can survive in the absence of IL-5 are isolated.

Isolation of the cDNA of Interest The cDNA expressed by this transduction vector is amplified, cloned and sequenced by PCR. A secondary screen is designed to test the ability of the gene to inhibit cell death associated with removal of IL-5 from eosinophils and TF-1 cells. Further studies are conducted to characterize the mode of action of the gene of interest and to identify possible uses of the gene in therapy (eg asthma) or other fields.

This approach may be useful for identifying directed immunosuppressive cDNAs or ribozymes for use in organ transplantation or other therapeutic areas. The identified nucleotides may be useful as an alternative to drugs that must be taken lifetime after organ transplantation.

This approach can be adapted to study positive or negative regulatory effects on any cellular transcription factor (another example is the family of transcription factor nuclear factor kappa B (NF-κB)).

Use of a lentiviral vector library to identify genes that affect the activation of transcription factor-dependent reporter genes. An example of this is looking for genes that prevent activation of activated T cell nuclear factor (NFAT) transcription factors.

Briefly, NFAT can be activated in vitro by stimulation of cells containing NFTA (such as human T cell lines) with calcium ionophores (such as ionomycin) and phorval esters (such as phorval myristyl acetate, PMA). This treatment activates calcineurin phosphatase (Ca ²⁺ dependent Ser / Thr protein phosphatase), which in turn dephosphorylates NFAT. Dephosphorylation of NFAT results in nuclear localization where it initiates transcription of NFAT-dependent genes along with the transcriptional coactivator AP-1 (activated by PMA treatment).

In order to detect cDNA that affects transcription of a gene that depends on the transcription factor of interest, it is necessary to obtain a cell line that expresses a reporter gene dependent on that factor. This can then be exploited using a lentiviral library for genes with positive or negative regulatory effects.

cDNA Library A cDNA library is prepared that expresses cDNA from various human immune cell types under the control of a constitutively active promoter.

Target host cell (including reporter gene)
Human T cell lines are stably transfected with an NFAT-dependent reporter gene such as GFP (LNGFR).

Vector Genome Design These cells are transduced with lentiviral vector cDNA libraries that express cDNA from various human immune cell types under the control of a constitutively active promoter.

Stimulation of host cells Transduced cells are exposed to stimuli that usually result in activation of the GFP reporter gene.

Cell screening Cells are sorted by FACS to remove cells that express high levels of GFP. These cells are grown under continuous stimulation and subjected to a further series of low level GFP expressing cells enriched by FACS.

Single cells are isolated and the cDNA expressed by the transduced virus is cloned, sequenced and identified. These genes are evaluated simultaneously with known inhibitors of related transcription factors (eg, cyclosporin A is an effective inhibitor of NFAT-dependent gene expression).

This approach may be useful for identifying directed immunosuppressive cDNAs for use in organ transplants or other therapeutic areas. This approach can be adapted to study positive or negative regulatory effects on any cellular transcription factor (another example is the family of transcription factor nuclear factor kappa B (NF-κB)). In order to detect cDNA that affects transcription of a gene that depends on the transcription factor of interest, it is necessary to obtain a cell line that expresses a reporter gene dependent on that factor. This can then be exploited using a lentiviral library for genes with positive or negative regulatory effects.

Another possibility is to use a lentiviral ribozyme library. This is used to isolate negative artificial regulators of transcription factors.

Chronic myelogenous leukemia (CML): Removal of Bcr-Abl tyrosine kinase CML is a hematopoietic malignancy originating from stem cells. This is associated with a specific cytogenetic damage, the Philadelphia chromosomal translocation. The genomic sequence in the BCR gene on chromosome 22 is in parallel with the genomic sequence of the ABL tyrosine kinase gene on chromosome 9. This results in the exchange of the sequence encoded by the first exon of ABL with the BCR-inducible sequence and expression of the 210 Kd chimeric fusion protein Bcr-Abl with unregulated kinase activity. In some acute lymphoblastic leukemias (ALL), a 185 kDa chimeric protein product is formed, reflecting another BCR rearrangement site.

Expression of this protein results in cytoskeletal and adhesion abnormalities resulting in proliferative and anti-apoptotic signals through the activity of the RAS, PI3 'kinase, STAT, ERK, and JNK MAP kinase pathways. This is retained exclusively in the cytoplasm of the transformed cells and has been reported to induce apoptosis when trapped in the nucleus (Vigneri and Wang, 2001, Nat Med. February 2001, 7 (2): 228-34).

The drug STI-571 (Gleevec), a tyrosine kinase inhibitor, has been shown to be very effective in the treatment of CML in the early chronic phase by binding to p210 and switching off. However, in a later acute phase, “acute transformation” due to further genomic instability, the patient initially responds well to the drug but mostly relapses. This is the result of a single mutation in p210 or an increase in the number of BCR-ABL genes that resulted in acutely transformed cells becoming resistant to STI-571 (Gorre et al., 2001, Science, August 3, 2001, 293 (5531): 876-80). Therefore, it is believed that drug cocktails may be important for the treatment of this stage of CML (McCormick, 2001, Nature, July 19, 2001, 412 (6844): 281-2). The fact that Bcr-Abl remains active in STI-571 resistant cells suggests that this chimeric oncoprotein is still a reasonable drug target and that expression of the Bcr-Abl oncogene is at the translational level. Several therapies that are inhibited have been presented (Jahagidar et al., 2001, Exp Hematol, May 2001, 29 (5): 543-56).

Experimental Method One approach is to use those promoters to drive reporter expression to identify genes whose transcription is upregulated by Bcr-Abl and to identify downstream effectors of the fusion protein. However, the disadvantage of this method is that tyrosine kinases are likely to have many substrates, such as direct phosphorylation of STAT5 (Sillaber et al., 2000, Blood, March 15, 2000, 95 (6): 2118- 25) It is difficult to target all pathways activated by Bcr-Abl. Furthermore, these signaling pathways are likely to be important for normal cell function.

A better method is to inhibit the expression of the Bcr-Abl oncogene itself. Transcription is reported to be regulated under the BCR promoter and its function remains intact despite genomic rearrangements. This further has the effect of inhibiting the expression of the Bcr protein encoded by the normal allele and the function of serine / threonine kinases is not known (Liu et al., 1996, Mol Cell Biol. March 1996). 16 (3): 998-1005) have been reported to antagonize the oncogene action of Bcr-Abl (Arlinghaus, 1998, Crit Rev Oncog, 1998; 9 (1): 1-18). This BCR promoter was functionally localized in the 1 kb 5 'region of the BCR exon 1 coding sequence (Shah et al., 1991, Mol Cell Biol, April 1991, 11 (4): 1854-60, Zhu et al. 1990, Nucleic Acids Res, December 11, 1990, 18 (23): 7119-7125), however, low steady state levels of BCR mRNA have been reported. Therefore, the use of LNGFR rather than GFP may be advantageous for improved detection of reporter gene products.

Bcr is evolutionarily conserved and has been reported to be expressed in many different types of human cells (Collins et al., 1987, Mol Cell Biol, August 1987, 7 (8): 2870-2876. Therefore, it should be considered that the removal of BCR expression may be toxic to normal cells. Nevertheless, short-term treatment with an inhibitor of BCR expression in combination with STI-571 may be sufficient to provide an effective treatment. Importantly, BCR is not an essential gene (Voncken et al., 1998, Int J Mol Med, November 1998, 2 (5): 577-583), and Bcr null mutant mice have neutrophil respiratory burst It has been reported to show an increase, as well as hormonal and behavioral stress responses but normal development and fertility (Voncken et al., 1995, Cell, March 10, 1995, 80 (5): 719-728. 1998, Oncogene, April 16, 1998, 16 (15): 2029-2032). Analysis of new CML and normal hematopoietic bone marrow cells reports that p210 and normal Bcr and AbI proteins are expressed primarily in the early steps of bone marrow maturation, and the level of expression is significantly reduced when cells mature into polymorphonuclear leukocytes. (Wetzler et al., 1993, J Clin Invest, October 1993, 92 (4): 1925-1939).

A therapeutic method that combines an agent that lowers Bcr-Abl levels, such as arsenic trioxide (As2O3), with an agent that inhibits Bcr-AblTK activity, such as STI-571, is responsible for apoptosis and differentiation of Bcr-Abl positive human leukemia cells This approach is confirmed by data demonstrating strong induction (Perkins et al., 2000, Blood, February 1, 2000, 95 (3): 1014-22, Porosnic et al., 2001, Leukemia, 2001 5 Moon, 15 (5): 772-778). Indeed, prior to chemotherapy, arsenic derivatives were used for the treatment of chronic myeloid leukemia (Kwong and Todd, 1997, Blood, May 1, 1997, 89 (9): 3487-8, Puccitti. Et al., 2000, Cancer Res, July 1, 2000, 60 (13): 3409-3413).

Studies of the expression level of Bcr-Abl transcription in chronic and blast phase cells have shown that, in the absence of genome amplification, occasionally an increase in steady state levels occurs in blast phase cells. This is due to changes in BCR promoter activity (Andrews and Collins, 1987, Leukemia, October 1987, 1 (10): 718-24), indicating the importance of identifying elements that control BCR expression. ing.

Outline of Experiment 1. Construction of a lentiviral vector containing the LNGFR reporter gene cassette under the control of the BCR promoter 2. Test constructs in Philadelphia chromosome positive and negative cell lines 3. Establish a stable cell line or pool containing the reporter construct. 4. Transduction of reporter cells with lentiviral ribozyme library (containing antibiotic resistance marker) 5. Isolate transduced cells by antibiotic selection 6. Remove LNGFR expressing cells using MAC beads 7. Isolate library construct from remaining cells for retesting Another approach to identify elements that up-regulate further downstream testing and confirmation expression involves screening of cDNA libraries (Gateway ™ compatible vector APh + CML libraries are available).

The polycystic kidney PKD1 gene is the most common human hereditary disease with a frequency of 1: 400 to 1: 1000 and is responsible for almost 10% of adult cases of end-stage renal failure. It accounts for 85% of cystic kidney (ADPKD) (Dalgaard, 1957, Acta Med Scand, 1957, 328: 1-255). The disease is generally late (30 to 70 years) and is mainly characterized by the formation of fluid-filled cysts in the kidneys.

Renal cysts most often occur after a second somatic event that inactivates the inherited healthy allele of the same gene or the corresponding gene. The 2.5 kilobase versus poly (purine pyrimidine) (poly (RY)) region present in intron 21 of the polycystic kidney 1 (PKD1) gene can stimulate repair and / or recombination functions Have been reported to cause high gene mutation frequency (Bacolla et al., 2001, J Biol Chem, May 25, 2001, 276 (21): 18597-604). However, the essentially high frequency of somatic second hits in the epithelium appears to be sufficient to explain somatic second hits that occur frequently in pathogenic genes (Arnaout, 2001, Annu Rev Med, 2001, 52: 93- 123, Review).

About half of people with a major type of PKD progress to renal failure, ie end-stage renal disease (ESRD) in which the patient requires dialysis or kidney transplantation.

The same clinical phenotype in human patients and targeted Pkd1 and Pkd2 mutant mouse models provides evidence that both gene products act in the same pathogenic pathway. The putative protein domain structure of polycystin 1 suggests that polycystin 1 is involved in cell-cell or cell-matrix interactions, and the similarity of polycystin 2 and polycystin L to the pore-forming domain of some cation channels Suggest that they form subunits of large plasma membrane ion channels. This PKD protein is capable of initiating signal transduction and activation of several downstream effectors including heterotrimeric G protein, protein kinase C, mitogen activated protein kinase, beta-catenin, and AP-1 transcription factor Bring. Furthermore, polycystin 2 may function in mediating calcium flux.
FIG. 4 shows two components of the proposed polycystin signaling pathway.

The pathogenesis of cyst formation is currently thought to include increased cell proliferation, fluid retention, and basement membrane remodeling. Cyclic adenosine monophosphate (cAMP) metabolism is a major component of cyst formation and is thought to stimulate apical chloride secretion by the CFTR channel and drive cyst fluid retention. In contrast to normal kidney cells, where cell proliferation is inhibited by cyclic AMP, ADPKD cells are stimulated to proliferate. Thus, alteration of polycystin function can transform normal cell phenotype into a phenotype that responds to increased cyclic AMP by increasing cell growth rate, and enlarged cysts can become larger by increasing the rate of cyclic AMP-driven fluid secretion High nature. Growth factors, including cyclic AMP and epidermal growth factor, have complementary effects that promote ADPKD cyst expansion, thereby contributing to disease progression. This knowledge should help to find inhibitors of the signaling cascade that can be used to treat ADPKD (Calvet and Grantham, 2001, Semin Nephrol, March 2001, 21 (2): 107-23, Review. ).

Increased cell proliferation and apoptosis have been implicated in the pathogenesis of cystic disease. Boletta et al. (2000, Mol Cell, November 2000, 6 (5): 1267-73), PKD1 functions to regulate both pathways and may send cells to differentiation pathways leading to tubule formation. It suggests that there is. In a recent review, Grantham (2001, Curr Opin Nephrol Hypertens, July 2001, 10 (4): 533-42) controls epithelial morphogenesis and maturation, a signal whose disruption is central to the cyst problem. We anticipate that the fields of physiology and molecular biology will be gathered for the transmission pathway. Arnold et al. (1999, Mol Cell Biol, May 1999, 19 (5): 3423-34) found that PKD2 up-regulates AP-1-dependent transcription in human fetal kidney 293T cells. This PKD2-mediated AP-1 activity was dependent on the activation of mitogen-activated protein kinase p38, JNK1, protein kinase C (PKC) epsilon, a calcium-independent PKC isozyme.

Co-expression of PKD2 with the interacting C-terminus of PKD1 dramatically increased PKD2-mediated AP-1 activation. AP-1 is a transcription factor that regulates various cellular programs such as proliferation, differentiation, and apoptosis. Activation of these signaling cascades may promote full maturation of developing tubular epithelial cells, and inactivation of these signaling cascades prevents terminal differentiation and prevents tubular cyst development. There is a possibility to promote.

At the cellular level, PKD mutations result in an abnormal distribution of epidermal growth factor receptor (EGFR) in the tubule epithelium. This receptor is usually restricted to the basal lateral region of the tubule epithelium but appears on the tip (lumen) surface in the cyst epithelium. Since cyst fluid contains epidermal growth factor and EGFR binds ligand and transmits mitogenic signals, improper localization of EGFR may be an important mechanism driving cyst growth. In organ culture, cyst formation can be prevented by blocking EGFR activity using an anti-EGFR antibody or tyrophostin that inhibits the tyrosine kinase activity of the receptor (Pugh et al., 1995, Kidney Int. March 1995). 47 (3): 774-81).

In Tg737-deficient mice (a model of multiple cystic kidneys), introduction of inactive EGFR (which blocks the activity of improperly localized EGFR) into the cyst epithelium substantially reduces cyst formation (Richard). Et al., 1998, J Clin Invest, March 1, 1998, 101 (5): 935-9). Future treatments for polycystic kidney disease include gene therapy or using tylophostin to inhibit the activity of improperly localized EGFR, or antibodies or other neutralizing agents to reduce epithelial growth factor in urine. May be directed to neutralize.

Experimental method 1. Confirmation of PKD1 expression in A431 cells (Northern)
2. Deletion by homologous recombination (2 rounds)
3. 3. Knockout gene expression versus control array profiling 4. Short list of genes whose expression / repression requires PKD1 5. Select candidate promoter to drive reporter gene expression 6. Build stable cell line using promoter / reporter cassette 7. Transduction with lentiviral library Screening for reporter block / switch on

PKD1 is highly expressed in fetal kidney (Ibragimov-Beskrovnaya et al., 1997, Proc Natl Acad Sci USA, June 10, 1997, 94 (12): 6397-402), and mouse homozygous mutant PKD1 alleles are embryonic lethal (Kim, 2000, Proc Natl Acad Sci USA, February 15, 2000, 97 (4): 1731-6). Polycystin 1 is detected in epithelial cells, astrocytes, endothelial cells, and vascular smooth muscle cells. Use of additional surrogate cell types is useful to determine whether the pathways induced by PKD1 signaling are common or specific to tissue types known to require proper functioning of PKD1 Will.

Selection of candidates that drive reporter gene expression should take into account that only a single branch of the signal transduction pathway activated by PKD1 can be targeted. Thus, where practical, selection of additional candidates and parallel screening with another reporter construct would be advantageous.

There are mouse and rat models of PKD, and in vitro models of cyst formation (Pey et al., 1999, In Vitro Cell Dev Biol Anim, November-December 1999, 35 (10): 571-9), further downstream Facilitates testing.

Use of lentiviral vector library to identify genes that affect the activation of the NFAT family of transcription factors Activated T-cells (NFAT) is the first human A family of transcription factors identified in T cells. NFAT consists of five members (current), and the four most characteristic (NFAT 1-4) are regulated by the ubiquitously expressed Ca ²⁺ -dependent Ser / Thr protein phosphatase, calcineurin (CaN). NFAT can be activated in vitro by stimulating with calcium ionophores (eg, ionomycin) and phorbal esters (PMA that non-specifically activate PKC). CaN is a major target for the immunosuppressant drug, cyclosporin A (CsA). CsA acts by first binding to the immunophilin ligand cyclophilin A (CypA) and then this drug-immunophilin complex binds to CaN phosphatase and inactivates its activity. By inhibition of CaN, CsA prevents CaN-mediated activation of NFAT and therefore NFAT-dependent gene transcription. Inactive NFAT is phosphorylated in the cytoplasm of resting T cells, and when CaN is activated by the release of intracellular Ca ²⁺ , NFAT is dephosphorylated by CaN. This results in exposure of the nuclear localization signal (NLS), and NFAT translocates to the nucleus. Once in the nucleus, NFAT works with the transcriptional coactivator AP-1 (activated by the PKC pathway) to initiate transcription of genes that contain NFAT elements in their promoter-enhancer regulatory domain. CsA is the current first choice in the multi-billion dollar organ transplant market. Another immunosuppressive drug (FK506) acts in a similar manner by binding to immunophilin FKBP12 and inactivating CaN phosphatase activity. Rapamycin is an alternative drug that inhibits NFAT as well, despite other mechanisms. The regulation and function of NFAT is described in Rao et al., Annu. Rev. Immunol. 15, 707-747.

There is a need for more specific means of immunosuppression. CsA has a wide range of serious side effects, including renal failure and various cancers. Because of the nature of organ transplants, patients need to take CsA continuously throughout their lives, thus increasing the probability that such patients will have side effects. One approach to develop a more specific new mechanism of immunosuppression is by using a peptide containing the IXIT CaN binding motif (SPRIEIT) present in the CaN-regulated NFAT protein (Patent International Publication Number WO99 / 40930) . To date, no human gene has been shown to regulate CaN-mediated activation of NFAT by this CaN-binding motif. However, there is one protein that has been shown to utilize this motif to inhibit CaN phosphatase activity by binding CaN at the same site as NFAT. This is the A238L protein from African swine fever virus (Miskin et al., 1998 and 2000, Science, 281, 562-565, J. Virol. 74, 9412-9420). A238L has also been shown to inhibit NF-κB-dependent gene transcription (Powell et al., J. Virol. 70, 8527-33, Tait et al., J. Biol. Chem. 275, 34656-64).

Methods A human T cell line is engineered to contain an NFAT-dependent reporter gene such as GFP (or LNGFR). This is accomplished by transducing T cells (Alo-T cells or cell lines such as Jurkat). This reporter can take several different forms. One possibility is to control the expression of the reporter gene with an artificial NFAT promoter-enhancer region containing 3 tandem copies of the NFAT enhancer element upstream of the minimal CMV promoter. A second possibility is to clone the promoter of a known NFAT-dependent gene (such as IL-2 in CD4 + T cells). However, this is almost certainly more confusing because several other transcription factors are known to provide IL-2 transcriptional control. A further possibility is a promoter that can be silenced by NFAT-NFATc2 and have the silencer activity of the interleukin-12 (IL-12) receptor beta2 proximity promoter in human T helper cells. This reporter is validated with a known NFAT-dependent gene transcription inhibitor (eg, cyclosporin A is an effective inhibitor of NFAT-dependent gene expression) or ASFV A238L.

These cells are transduced with lentiviral vector cDNA libraries that express cDNA from various human immune cell types (such as T cells) under the control of a regulated promoter or a constitutively active promoter. Transduced cells are exposed to stimuli that usually result in activation of the GFP reporter gene (eg, PMA and ionomycin). To remove cells that express high levels of GFP, the cells are sorted by FACS (or vice versa if a silencing promoter is used). These cells are grown under continuous stimulation and subjected to a further series of low level GFP expressing cells enriched by FACS. Single cells are isolated and the cDNA expressed by the transduced virus is cloned, sequenced and identified. These genes are evaluated simultaneously with known inhibitors of related transcription factors. This approach may be useful for identifying directed immunosuppressive cDNAs for use in organ transplants or other therapeutic areas (eg, CsA is used for RA).

Another possibility is to use a lentiviral ribozyme library. This is used to isolate active ribozymes that inhibit NFAT activation or expression.

Ribozyme selection method The selection of ribozymes is performed in vivo (tissue culture). Ribozymes constructed and tested “in theory” in vitro often do not work well in a cellular environment.

In one embodiment, the present invention uses a multicistronic, preferably bicistronic approach.

In this embodiment, the target vector, which can be pONY8.4 based, is bicistronic and the expression of the tetracycline repressor is driven by the LTR. The target gene or sequence is cloned downstream of the tetracycline repressor, preferably no IRES is present, so no expression of the protein from the subject noi occurs. Transduced reporter cells can be selected by adding Dox. Dox binds to a tetracycline repressor, thereby derepressing the Tet operator and expressing LNGFR. The cells can then be selected by FACS, MACS, or other suitable method. After selection, Dox is removed and LNGFR is no longer expressed. A schematic of this target vector is shown in FIG.

This library vector (also pONY8.4 based) is also bicistronic. The expression of neo is driven by the LTR and is selected as needed. There are several genes downstream of this that are out of frame and / or do not contain IRES, which are not preferred for cleavage by ribozymes. These include tetracycline inhibitors, LNGFR, and GFP. If the library ribozyme cleaves any of these gene sequences, there is no packaged genome and the ribozyme should not be represented in the vector library used to transduce reporter cells. Furthermore, producer cells die when ribozymes cleave essential molecules (eg, RNA polymerase). Ribozyme expression is under the control of the CMV promoter. A schematic diagram of this library vector is shown in FIG.

Method Summary Reporter cells (containing the target vector) are transduced with a ribozyme library.
Cleavage of gene X results in degradation of mRNA encoding TetR.
The Tet operator is derepressed and LNGFR is expressed.

Cell Isolation Cells are labeled with α-LNGFR, incubated with appropriately conjugated MACS beads, and passed through a MACS column. This approach is illustrated in FIG.

Maintain LNGFR expressing cells. These can be subcultured to increase cell numbers (neo selection can be applied to ensure that only cells containing the library vector are present). FACS analysis can be performed to measure LNGFR expression levels. This ribozyme can be amplified and sequenced by PCR.

pONY8.4
The series of pONY8.4 vectors has several modifications that allow it to function as part of a transient or permanent vector system that is completely independent of accessory proteins without adversely affecting titer. Have Usually, to obtain an appropriate titer, the lentiviral vector genome required the presence of the viral protein rev in the producer cell (transient or permanent). This includes the current HIV vector system as well as previous EIAV vectors. There are four modifications compared to a series of pONY8.1 vector genomes. They are as follows.
a) All ATG motifs derived from gag and forming part of the packaging signal have been modified to read ATTG. This allows the insertion of an open reading frame that can be driven by the LTR promoter.
b) The length of the genome, i.e. the distance between the R regions, is close to that found in the wild type virus (7.9 kb).
c) The 3′U3 region has been modified to contain sequences from the Moloney leukemia virus (MLV) U3 region, so that when transduced, a second open reading frame (ORF) is added in addition to the internal cassette. Can be driven. In this example, Applicants use MLV, but can be any promoter.
d) The vector contains a nucleotide sequence operably linked to a viral LTR, the nucleotide sequence upstream of an internal promoter, and the nucleotide sequence encodes a polypeptide or fragment thereof.

All of these modifications allow the production of viral delivery systems without the need for accessory proteins, and target cells incorporate only 10% of the original viral sequence. These factors are important for future safety considerations in terms of immune response and the potential for production of replicating viruses. Further details regarding modification of the LTR can be found in Applicants' WO96 / 37623 and WO98 / 17816.

FIG. 7 is a schematic representation of the EIAV genome. These genomes can be used to transfect according to the method of the invention. Upon transfection, the 3 ′ LTR is copied to the 5 ′ LTR. FIG. 8 shows the entire plasmid sequence of pONY8.1G. FIG. 9 shows the entire plasmid sequence of pONY8.4ZCG. FIG. 10 shows the entire plasmid sequence of pONY8.4GCZ.
FIG. 11 is a schematic diagram of the hybrid U3 region.
FIG. 12 shows the sequence of the hybrid LTR.

Construction of EIAV / MLV hybrid LTR vector PCR was performed as follows.
Product A = Primer KM001 + KM003, pONY8.1Z as target
Product B = Primer KM004 + KM005, pHIT111 as target
Product C = Primer KM006 + KM002 with pONY8.1Z as target

PCR products (A, B, and C) were gel purified. Products A and B (with primers KM001 and KM005) were used to construct a PCR reaction and product D was obtained. Products B and C (with primers KM004 and KM002) were used to construct a PCR reaction and product E was obtained. Products D and E were gel purified and used in PCR reactions as targets with primers KM001 and KM002 to give product F. PCR product F was gel purified (about 1 kb). This was then cleaved with SapI and subcloned into pONY8.1Z cleaved with SapI. As a result, the vector pONY8.1Z hybrid shown in FIGS. 13 and 14 was obtained. The EIAV 3 'LTR was replaced with an EIAV / MLV hybrid LTR. EIAV U3 was almost replaced by the MLV U3 region. Since the EIAV5'U3 sequence of the 3'LTR contains an att site, which is the sequence required for integration, the EIAV5'U3 sequence of the 3'LTR was retained.

Primer sequences are shown below.
EIAV / MLV hybrid U3

Final PCR product sequence

Parkinson's Disease-Identification of Regulators of Tyrosine Hydroxylase Tyrosine hydroxylase (TH) is an enzyme required for dopamine synthesis. Nerve growth factor is known to induce TH in rat sympathetic ganglia, adrenal medulla, sympathetic neurons, and chromaffin cells.

A molecular biological development with therapeutic potential to treat Parkinson's disease is the cloning of genes encoding neurotrophins and growth factors. For example, glial-derived neurotrophic factor (GDNF) can stop the progression of nigrodegeneration induced by the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). It has been reported that GDNF infusion has been reported to reduce symptoms in a primate Parkinsonism model. Sonic hedgehog, a signaling molecule known to have an important role in several developmental situations, induces midbrain neurons to develop and differentiate into dopaminergic phenotypes in vitro Can do. These results suggest that a new therapeutic strategy may be to identify molecules that promote the differentiation or survival of dopaminergic neurons.

Another hypothesis is that dopaminergic neurons simply stop expressing genes involved in dopamine production. In response to GDNF, dopaminergic cells may actually activate the expression of these genes. In this case, the identification of molecules that activate the expression of these genes would be a therapeutic function to consider. Thus, these molecules provide the therapeutic effect seen with GDNF without the side effects of cell proliferation.

One approach to identify molecules that modulate the expression of tyrosine hydroxylase is as follows.
Method 1. The tyrosine hydroxylase promoter is cloned such that its activity is monitored by an appropriate reporter gene such as green fluorescent protein (GFP), low affinity growth factor receptor (LNGFR), or thymidine kinase (TK).
2. The promoter-reporter cassette is introduced into a primary neuron or dopaminergic cell line such as 1RB3AN27, which may or may not normally express tyrosine hydroxylase.
3. Reporter cells are transduced with an appropriate library of EIAV vectors (such as cDNA or ribozyme).
4). Optionally, the cells are grown in the presence of GDNF or other growth factor.
5). Cells with altered reporter gene expression are isolated and the modulating moiety is isolated from the library vector by sequencing.
6). The target portion is identified from the sequence of the library insert.

The relative REV dependence of pONY8.1ZCG and pONY8.0G was determined by evaluating the transduction efficiency in the presence and absence of REV (see FIG. 16). This was done using the pESSYNREV vector. pESYNREV and pONY8.0Z are described in WO0179518. pONY8.0G is derived from pONY8.0Z with lacZ replaced with GFP. pONY8.1ZCG is derived from pONY8.4ZCG but does not have a hybrid LTR or complete ATTG sequence. An outline of pONY8.1ZCG and pONY8.0G is shown in FIG. The introduction efficiency of pONY8.1ZCG and pONY8.0G was evaluated using a standard cell line in the presence and absence of REV.

Producer cell cytoplasmic RNA levels were measured using quantitative PCR and normalized to 100 copies of cellular β-actin. Measurements were made at the time of virus harvesting and show that the level of RNA with EIAV packaging signal is reduced in pONY8.0G producer cells in the absence of REV. This difference is not seen in pONY8.1ZCG producer cells. This data indicates that the pONY8.0G (± pESYNREV) construct is REV-dependent and the pONY8.1ZCG introduction efficiency is REV-independent.

Using the pONY8.4EIAV vector, a T-Rex tetracycline regulatory system was incorporated to create a tetracycline regulatory vector to produce pONY8.4TsynCoG1 (
19 and 20). This TetO ₂ promoter was codon optimized for expression in mammalian cells (FIG. 21). Expression was measured using GFP as a reporter gene. pONY8.4TsynCoG1 expression levels (FIG. 23) is coupled to TetR, compared to the absence of doxycycline (dox) for releasing the inhibition of the TetO _2, it was shown 10 times higher in the presence thereof. Comparison of GFP expression with parental plasmids pONY8.4ZCG and pONY8.4TCOG1 shows that suppression in the absence of dox is obtained only with pONY8.4ZTsynCOG1.

Cells transduced with the pONY8.4ZCG-derived vector were cultured in medium with or without dox, replaced to properly induce or suppress expression, and then returned again. The “reversibility” of expression of the Tet regulatory gene from the vector containing the codon optimized TetR was confirmed. As shown in FIG. 24, the GFP expression seen in the exchanged cells has not reached the highest / lowest level observed in cells that grew continuously at either + or −dox.

PONY8.4TsynnnsCOG1 was generated in which TetR contains the following nuclear localization signal (NLS) at its C-terminus.

NLS has been reported to increase transcriptional repression by a factor of 3 by increasing the concentration of TetR in the nucleus (Ogueta et al.). This NLS was ligated to the C-terminus so as not to interfere with the N-terminus of TetR, which is important for TetO ₂ binding.

To investigate how quickly expression occurs after dox addition, a time course was performed (reduction in expression after dox removal as measured by GFP is affected by protein turnover).
FIG. 24 shows that expression is almost maximal within 24 hours of dox addition and that the NLS-tagged codon optimized TetR has no lower basal expression compared to the unlabeled form.

FIG. 25 shows GFP expression from the surrogate LTR PGK and RSV of the pONY8.3 vector, demonstrating that the surrogate hybrid LTR can also be used in the pONY8.4 constituent vector.

The bicistronic Tet regulatory vector can be modified to incorporate a second copy of the Tet repressor gene (FIG. 26). This second copy is downstream of the IRES. This is actively expressed when cells are initially transduced and initially do not contain the TetR protein. As protein levels increase, transcription of genes X and TetR located downstream of the IRES stops (no tetracycline or doxycycline is present). This suppression is more rapid in the modified constructs shown here.

In the absence of LTR driven TetR, there is a feedback loop that results in a circulation of gene X transcription depending on the threshold of TetR. The presence of a constitutively transcribed copy of TetR avoids this by keeping the level of TetR in the cell constant. In this example, the TetR gene downstream of the IRES is a wild type that minimizes the possibility of unwanted recombination, however any combination of gene sequences encoding the TetR protein can be envisioned (NOI is also an IRES). Can be located downstream).

The expression from the pONY8.4 and pONY8.7 vectors was examined in the presence and absence of Rev by assaying the virus titer of the infected cells. Viral data shown is from non-concentrated, single plate. pONY8.7NCZ was tested at a different week than the other vector, and thus slightly higher titer. The absence of Rev did not make a significant difference in the titers of the pONY8.4 and pONY8.7 vectors, indicating that it was rev independent.

All publications mentioned in this specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described with reference to certain preferred embodiments, it is to be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are within the scope of the following claims.

1 is a schematic representation of the screening method of the present invention. 1 is a schematic representation of a library construct suitable for introduction of NOI into target cells. 1 is a schematic representation of a lentiviral method for identifying drug targets for inflammatory diseases. FIG. 2 shows two components of the presented polycystin signaling pathway. 1 is a schematic representation of a bicistronic targeting vector. 1 is a schematic representation of a bicistronic library vector. It is a figure which illustrates isolation of a cell. 1 is a schematic representation of the EIAV genome. FIG. 8 shows the entire plasmid sequence of pONY8.1G. FIG. 8 shows the entire plasmid sequence of pONY8.1G. FIG. 9 shows the entire plasmid sequence of pONY8.4ZCG. FIG. 9 shows the entire plasmid sequence of pONY8.4ZCG. FIG. 9 shows the entire plasmid sequence of pONY8.4ZCG. FIG. 10 shows the entire plasmid sequence of pONY8.4GCZ. FIG. 10 shows the entire plasmid sequence of pONY8.4GCZ. FIG. 10 shows the entire plasmid sequence of pONY8.4GCZ. 1 is a schematic diagram of a hybrid U3 region. It is a figure which shows the arrangement | sequence of hybrid LTR. FIG. 13 is a diagram showing the arrangement of pONY8.1Zhyb. FIG. 13 is a diagram showing the arrangement of pONY8.1Zhyb. FIG. 13 is a diagram showing the arrangement of pONY8.1Zhyb. Schematic representation of pONY8.1Zhyb. Schematic representation of pONY8.1ZCG and pONY8.0G. FIG. 6 is a graph showing the relative transduction efficiency of pONY8.1ZCG and pONY8.0G in the presence and absence of Rev. FIG. 17 shows the complete sequence of pONY8.4TCOG. FIG. 17 shows the complete sequence of pONY8.4TCOG. FIG. 17 shows the complete sequence of pONY8.4TCOG. FIG. 18 shows the complete sequence of pONY8.4TsynCOG / pONY8.4TsynCOG1. FIG. 18 shows the complete sequence of pONY8.4TsynCOG / pONY8.4TsynCOG1. FIG. 18 shows the complete sequence of pONY8.4TsynCOG / pONY8.4TsynCOG1. It is the schematic of pONY8.4TsynCOG / pONY8.4TsynCOG1. 1 is a schematic illustration of an example of a bicistronic tetracycline regulatory vector. FIG. 5 shows an analysis of codon usage of the TetR gene. MH = mammalian high expression gene, WT = wild type TetR gene, CO = codon optimized TetR gene. Codon usage has been modified to closely resemble high-expressing mammalian genes. FIG. 5 shows an alignment of wild type, codon optimized Tet repressor gene sequences. The sequence is 71% identical at the nucleotide level (longest consecutive 13 bp of homology). FIG. 5 shows an alignment of wild type, codon optimized Tet repressor gene sequences. The sequence is 71% identical at the nucleotide level (longest consecutive 13 bp of homology). FIG. 5 shows an alignment of wild type, codon optimized Tet repressor gene sequences. The sequence is 71% identical at the nucleotide level (longest consecutive 13 bp of homology). It is a figure which shows that GFP expression in the absence of dox decreases 10 times from coexistence of dox by codon optimization TetR. This difference is significantly greater than when using other vectors, suggesting that pONY8.4TsynCOG allows better control of suppression. FIG. 5 shows reversibility of Tet regulatory gene expression from vectors containing codon optimized TetR. It is a figure which shows the time-dependent change of GFP expression after a doxycycline addition. It is a figure which shows the expression from alternative LTR. Figure 2 is a schematic representation of a modification of a bicistronic Tet regulatory vector that incorporates a second copy of a Tet repressor gene. FIG. 4 shows the virus titers of pONY8.4 and pONY8.7 vectors in the presence and absence of Rev. Expression of pONY8.4 and pONY8.7 is Rev independent.

Claims (33)

Downstream of the viral LTR, and includes a heterologous open reading frame (ORF) or a portion thereof upstream of the promoter, multicistronic lentiviral controlling the expression of one Downstream N O I even without the least said promoter Vector genome. 2. The vector of claim 1, wherein the heterologous ORF is operably linked to the LTR. The nucleic acid sequence encoding the auxiliary gene rev or its functional equivalent has been disrupted such that the auxiliary gene cannot encode a functional auxiliary protein, or has been excluded from the vector genome and not supplied in trans The vector according to claim 1 or 2. The vector according to any one of claims 1 to 3, wherein the heterologous ORF is a reporter moiety or a selectable marker. Heterologous OR F encodes a modulator gene vector according to any one of claims 1 to 3. The vector according to claim 5, wherein the modulator gene is selected from a tetracycline inhibitor such as TetR and a tetracycline-regulated transactivator such as tTA or rtTA . The lentiviral vector according to claim 6 , wherein the tetracycline inhibitor is codon optimized for expression in mammalian cells. The lentiviral vector according to any one of claims 1 to 7 , wherein the vector is a non-primate lentiviral vector. Vector, HIV-1, HIV-2 , SIV, FIV, BIV, EIAV, CEV, CAEV, MVV, or is selected from the group consisting of visna lentivirus, lentivirus according to one of claims 1 to 7 vector. Vectors comprises central polypurine tract (cPPT) sequence, lentiviral vector according to any of claims 1-9. Lentiviral vector according to any one of claims 1 to 10 including vectors are posttranscriptional regulatory element or a translational element. The lentiviral vector according to any one of claims 1 to 11 , wherein the vector comprises a gag packaging signal having an ATG motif, and the ATG motif is an ATTG motif. The lentiviral vector according to any one of claims 1 to 12, wherein the vector comprises a 3'U3 region having a viral promoter or eukaryotic promoter sequence. The lentiviral vector according to claim 13, wherein the viral promoter or eukaryotic promoter is a wild type promoter. The lentiviral vector according to claim 14 , wherein the viral promoter is an EIAV or MLV U3 region. The lentiviral vector according to any one of claims 1 to 15 , wherein the lentiviral vector is a self-inactivating lentiviral vector. Vector genome, 2, 3 or 4 cistronic vector according to any of claims 1-16. System includes a set of vector genome, Gag and Pol proteins, and env protein or nucleic acid sequence encoding a component of the vector containing those functional substitute according to any one of claims 1 to 17 Wrench A lentiviral vector production system for producing virus-derived vector particles. 19. The production system of claim 18, wherein the set of nucleic acid sequences comprises three DNA constructs encoding (i) vector genome, (ii) Gag and Pol, and (iii) Env. 19. A nucleic acid sequence encoding an auxiliary gene rev or functional alternative has been disrupted such that the auxiliary gene cannot encode a functional auxiliary protein, or has been removed from the vector genome and is not supplied in trans. Or the production system of 19. Auxiliary genes vpr retroviral derived particle child, vif, tat, and nef, or even a nucleic acid sequence encoding at least one of the similar auxiliary genes, the auxiliary gene is destroyed so that it can not encode a functional auxiliary protein dolphin or production system according to any one of claims 18 to 20 which is removed from the system. The production system according to any one of claims 18 to 21, wherein the nucleic acid sequences encoding Gag and Pol are codon optimized. 23. A DNA construct for use in a system according to any of claims 18-22, encoding a packageable RNA vector genome according to any of claims 1-17. A set of DNA constructs comprising a DNA construct according to claim 23 and a DNA construct encoding Gag and Pol according to any of claims 19-22. En v or set of D NA construct of claim 20 further comprising a DNA construct encoding a functional substitute. The method comprising the set of set or DNA construct nucleic acid sequence according to any of claims 23 to 25 into a host cell, method for preparing the retroviral vector particles which include the steps of obtaining a retroviral vector particles. A retroviral vector particle produced by the system or method according to any one of claims 18 to 26 . A retrovirus- derived vector particle comprising the RNA vector according to any one of claims 1 to 17 , a Gag and Pol protein, and an Env protein, or a functional substitute thereof. A cell transduced with the retroviral vector particle according to claim 27 or 28 , or the DNA construct according to any of claims 23 to 25 . 30. The retroviral vector particle according to claim 27 or 28 , the DNA construct according to any of claims 23 to 25 , or the cell according to claim 29 , which is used for medical treatment. Together with a pharmaceutically acceptable carrier or diluent, vector according to any one of claims 1 to 17 A system according to any one of claims 18 to 22, the particle element according to claim 27 or 28, or 30. A pharmaceutical composition comprising the cell of claim 29 . For the preparation of a medicament for delivering a NOI to a target site that require, retroviral vector particle according to claim 27 or 28, or claim 23 to 25 DNA construct according to any one, or to claim 29 Use of the described cell. 30. A delivery system in the form of a retroviral vector particle according to claim 27 or 28 , a DNA construct according to any of claims 23 to 25 , or a cell according to claim 29 for use in medicine.